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Lysosomal processing of sialoglycoconjugates in a wheat germ agglutinin resistant variant of EL4 murine leukemia cells
Devino, N aney Lynn, Ph.D.
The Florida State University, 1989
U·M·I 300 N. Zeeb Rd Ann Arbor, MI 48106
THE FLORIDA STATE UNIVERSITY
COLLEGE OF ARTS AND SCIENCES
LYSOSOMAL PROCESSING OF SIALOGLYCOCONJUGATES
IN A WHEAT GERM AGGLUTININ RESISTANT VARIANT
OF EL4 MURINE LEUKEMIA CELLS
By
NANCY L. DEVINO
A Dissertation submitted to the Department of Chemistry in partial
fulfillment of the requirements for the degree of Doctor of Philosophy
Degree Awarded:
Fall Semester, 1989
The members of the Committee approve the dissertation of
Nancy L. Devino defended on August 10, 1989.
or:: ~ Gilmer Professor Directing Dissertation
Dr. T. C. Keller Outside Committee Member
Dr. E. F. Hilinski Committee Member
ABSTRACT
Metabolic studies were undertaken in EL4 murine leukemia cells and
in WBG, a wheat germ agglutinin-resistant variant of EL4, in order to
- identify any differences in lysosomal processing of sialoglyco
conjugates. Five lysosomal acid hydrolases, acetylesterase, acid
phosphatase, (3 -galactosidase, ot -mannosidase, and neuraminidase, were
studied using fluorescent 4-methylumbelliferyl substrates. No
significant differences were found in the total activity of any of
these enzymes in EL4 and WB6. Cells were incubated in the presence of
N-acetylmannosamine, the metabolic precursor of sialic acid (N
acetylneuraminic acid). Free sialic acid accumulated in the lysosomes
of WBG but not of EL4. The accumulation of lysosomal free sialic acid
in WBG showed a dependence on the concentration of N-acetylmannosamine
in the growth medium. Metabolic labelling with [6- 3Hj-N-acetyl
mannosamine showed that WB6 accumulated lysosomal free sialic acid even
at very low concentrations of N-acetylmannosamine. The two cell lines
differed in their distribution of radiolabelled neutral sugars, free
sialic acid, and sialoglycoproteins. The velocity of (3H)-sialic acid
release was 3.7-fold lower in WB6 than in EL4, suggesting that WB6 has
a defect in lysosomal sialic acid transport. The metabolic
consequences of this defect are examined, in light of other biochemical
and immunological data on these cells.
iii
ACKNOWLEGEMENTS
I would like to thank Dr. Penny Gilmer for her support and
encouragement during this investigation. Special thanks to a few of my
closest friends for being there when things got rough: Duane, Roy,
Andrea, Kathy, and Diane. My fellow lab students Chris "Pete" Harrison
and Wayne "Fern" Baker deserve a hand for helping me see the humor in
our work. I also appreciate the helpful conversations with professors
Ed Hilinski, Tom Keller, and William Marzluff (FSU) and Ajit Varki
(UCSD). Jim Norris of the FSU Statistics Consulting Center deserves
special mention for his patient tutoring in the analysis of variance.
Finally, I would like to thank the FSU Department of Chemistry for
funding this project, especially in the spring and summer of 1989.
iv
TABLE OF CONTENTS ----
LIST OF TABLES x
LIST OF FIGURES xii
LIST OF ABBREVIATIONS xiv
Chapter One - Introduction
Section
LA. Biochemistry of Glycoconjugates 1
LA. 1. Oligosaccharide Biosynthesis 1
I.A.2. Biochemistry of Sialic Acids 12
I.A.3. Protein Glycosylation 17
I.A.4. Lectins and Their Uses 20
LB. Functions of Glycoprotein Carbohydrates 25
I.B.l. Non-specific Functions 26
I.B.2. Specific Functions 32
I.B.3. Carbohydrates and the Immune System 33
I.B.4. Functions of Sialic Acid 38
I.C. Lysosomal Processing of Glycoproteins 51
I.C.l. Biology of Lysosomes 51
I.C.2. Glycoprotein Degradation 52
v
I.C.3.
I.C. 3 .a.
I.C.3.b.
I.C.3.c.
I.D.
Section
II.A.
II.A.l.
II.A.2.
II.A.3.
II.B.
II.B.l.
II.B. 2.
II.C.
II.C.l.
II.C.2.
II.C.2.a.
II.C. 2 .b.
II.C.2.c.
II.C. 2 .d.
II.D.
II.E.
Defects in Lysosomal Transport
Cystinosis
Type C Niemann-Pick Disease
Sialic Acid Storage Disease
Overview of this Research
Chapter ~ - Materials and Methods
General Methods
Reagents and Equipment
Cell Lines
Protein Assay
Cell Fractionation in RSB
Cell Disruption by N2 Cavitation
Fractionation of Cells Disrupted by N2 Cavitation
Assays Utilizing Fluorescent Substrates
Fluorescence Measurements
Enzyme Assays
Neuraminidase Activity
f3 -Galactos idase and ~-Mannosidase Activity
Acid Phosphatase Activity
Acetylesterase Activity
Lysosomal Sensitivity to NaCl
Purification of Lysosomes in Isotonic Sucrose
vi
54
54
57
59
68
70
70
70
71
71
71
72
75
75
75
79
79
79
80
80
81
ILF.
II.G.
IIoG.l.
IIoG.2.
II.G.3.
IIoG.4.
IIoG.5.
Section
IIIoA.
IIIoB.
IIIoB.l.
IIIoB.2.
IIIoC.
IIIoC.l.
IIIoC.2
IIIoC.3.
III.C.3.a.
IIIoC.3.b.
IIIoC.4.
IIIoC.5.
Lysosomal Membrane Phosopholipid and Protein Analysis
Lysosomal Transport Studies
Determination of Lysosomal Sialic Acid
Distribution of Radioactivity Following [6-3H]-ManNAc Incubation
Lysosomal Stability as a Function of Time
Release of 3H Sialic Acid from Lysosomes
Accumulation of Lysosomal Cystine
Chapter Three - Results
In Vitro Cell Growth
Preliminary 4-MU Studies
4-MU Fluorescence as a Function of pH and Temperature
Design of Hydrolase Activity Experiments
studies on RSB-Prepared "Lysosomes"
Distribution of Lysosomal Activity
Lysosomal Sensitivity to [NaCl]
Neuraminidase Assays
Results
Statistical Analysis of Neuraminidase Data
.8-Galactosidase Activity
O(-Mannosidase Activity
vii
---------- ---- _._-- ----
84
85
85
88
89
89
90
94
95
95
101
101
101
104
110
110
112
113
115
IILD.
IILD.l.
IILD.2.
IILD.3.
IILD.4.
IILD.5.
IILD.6.
IILE.
IILE.l.
IILE.2.
IILE.3.
IILE.4.
IILE.5.
IILE.6.
Section
IV.A.
IV.B.
IV.B.l.
IV.B.2.
IV.C.
IV.C.l.
Studies on Percoll-Prepared Lysosomes
Purification of Lysosomes on Percoll Gradients
Neuraminidase Activity
t3-Galactosidase Activity
Acetylesterase Activity
Acid Phosphatase Activity
Analysis of Lysosomal Membrane Phospholipid and Protein
Lysosomal Transport Studies
Lysosomal Accumulation of Sialic Acid
WB6 Lysosomal Sialic Acid Accumulation in Varied [ManNAc]
Cellular Distribution of Radioactivity Using [6-3H]-ManNAc
Lysosomal Stability as a Function of Time
Velocity of Lysosomal Release
Lysosomal Cystine Accumulation
Chapter Four - Discussion
Summary
Lysosomal Transport Studies
Cold ManNAc Experiments
3H-ManNAc Studies
Acid Hydrolase Studies
Glycosidase Activities
viii
-------- -- ---_._._-- -----_._------- ----_ ..
115
115
119
122
124
124
127
129
129
133
137
149
152
156
159
160
160
164
170
170
IV.C.2.
IV.C.3.
IV.D.
IV.E.
REFERENCES
Acid Phosphatase Activity
Acetylesterase Activity
Lysosomal Membrane Phospholipid and Protein Studies
A Model for Altered Sialoglycoconjugate Metabolism in WB6 Cells
ix
173
175
176
177
182
Number
1-1
1-2
2-1
3-1
3-2
3-3
3-4
List of Tables
Title
Characterization of WGA-Resistant Cell Lines
9-0-Acetylation of Sialic Acids from EL4 and WB6 PM
Reagents and Suppliers
Dependence of 4-MU Fluorescence on pH
Distribution of F3-Galactosidase Activity in Cells Fractionated in RSB
Sensitivity of Lysosomes to [NaCI]
Neuraminidase Activity in RSB-Prepared "Lysosomes"
43
50
91
98
105
107
111
3-5 ~ -Galactosidase Activity in RSB-Prepared Cell Lysates 114
3-6
3-7
3-8
3-9
3-10
3-11
3-12
3-13
o(-Mannosidase Activity in RSB-Prepared Cell Lysates
Enhancement of ~-Galactosidase Specific Activity During EL4 Lysosome Purification
Neuraminidase Activity in LIB-Prepared Lysosomes from EL4 and WB6
Total p -Galactosi~ase Activity in LIB-Prepared Cell Lysates of EL4 and WB6
Total Acetylesterase Activity in LIB-Prepared Cell Lysates of EL4 and WB6
Total Acid Phosphatase Activity in LIB-Prepared Cell Lysates of EL4 and WB6
Analysis of Lysosomal Membrane Phospholipid and Protein
Lysosomal Accumulation of Sialic Acid in EL4 and WB6
x
116
120
121
123
125
126
128
130
3-14 Relative Lysosomal Accumulation of Sialic Acid 132
3-15 WB6 Lysosomal Sialic Acid Accumulation as a 134 Function of ManNAc Concentration
3-16 Distribution of Radioactivity in Lysosomal and 139 Cytoplasmic Fractions of EL4 and WB6
3-17 Distribution of Total Radioactivity in Lysosomal 142 and Cytoplasmic Fractions
3-18 Percent Distribution of Radioactivity in Lysosomal 144 and Cytoplasmic Fractions
3-19 Velocity of Lysosomal Sialic Acid Release 153
3-20 Lysosomal Cystine Accumulation 157
4-1 Comparison of EL4 and WB6 Lysosomal Free Sia/Unit 162 Acid P-ase in the Presence Or Absence of 50 roM ManNAc
4-2 Comparison of EL4/WB6 Activity Ratio for Three 174 Lysosomal Enzymes
xi
Number
1-1
1-2
1-3
1-4
1-5
1-6
1-7
1-8
1-9
1-10
1-11
2-1
2-2
2-3
2-4
3-1
3-2
3-3
3-4
List of Figures
Title
N-Linked High Mannose Oligosaccharides
Synthesis of N-Linked Oligosaccharide Core
En Bloc Transfer of Core Oligosaccharide to Protein
Post-Translational Modification of Oligosaccharides
General structure of Sialic Acid
Metabolism of Sialic Acids
Alteration of Galactosyltransferase Specificity by o(-Lactalburnin
Syngeneic Tumor Cell Growth
WB6 Growth and Viability in WGA
Synthesis of N-acetylneuraminic Acid
Metabolic Pathways of N-acetylrnannosamine
Cell Fractionation in RSB
Enzyme Assays utilizing 4-MU Substrates
Purification of Lysosomes on Percoll Gradients
Purification of Lysosomal Sialic Acid
Ionization of 4-Methylumbelliferone
pH Depende'lce of 4-MU Fluorescence
Representative Neuraminidase Assay
Sensitivity of Lysosomes to [NaCl]
xii
3
5
7
10
13
15
28
44
46
62
64
73
76
82
86
96
99
102
108
3-5 Distribution of EL4 ~-Galactosidase Activity 117 in LIB-Prepared Cells
3-6 WB6 Lysosomal Sialic Acid Accumulation 135
3-7 Cellular Distribution of Radioactivity in EL4 and WBG 140
3-8 Percent Distribution of Radioactivity in EL4 and WBG 145
3-9 Comparison of Percent Distribution of Radioactivity 147 in EL4 and WBG
3-10 Lysosomal stability at 370 C 150
3-11 Velocity of Lysosomal Sia Release 154
xiii
Ab
Ag
Asn
ATP
BSA
C
COME
CHO
CMP
Con A
CP
CPM
CTL
DMEM
OolP
DolPP
DTH
EGF
ER
Fuc
Gal
GalNAc
GOP
antibodies
antigens
asparagine
~ Q[ ABBREVIATIONS
adenosine triphosphate
bovine serum albumin
complement
cystine dimethyl ester
chinese hamster ovary
cytidine monophosphate
concanavalin A
Clostridium perfrinqens
counts per minute
cytotoxic T-lymphocyte
Dulbecco's Modified Eagle's Medium
dolichol phosphate
dolichol pyrophosphate
delayed-type hypersensitivity
epidermal growth factor
endoplasmic reticulum
fucose
galactose
N-acetylgalactosamine
guanosine diphosphate
xiv
Glc
GlcNAc
gly-carb
GTP
HexNAc
HS
Ig
IL-l
IL-2
IVC
LDL
LIB
L-PHA
M6P
Man
ManNAc
mcAb
Mg/ATP
MHC
MPR
4-MU
4-MU-Ac
4-MU-Gal
4-MU-Man
4-MU-P
glucose
N-acetylglucosamine
85 mM glycine-carbonate, pH 9.4
guanosine triphosphate
N-acetylhexosamine
heat-inactivated horse serum
immunoglobulin
interleukin 1
interleukin 2
Influenza virus C
low density lipoprotein
lysosome isolation buffer
leukophytohemagglutinin
mannose-6-phosphate
mannose
N-acetylmannosamine
monoclonal antibody
5 mM MgC12 and 2 mM adenosine triphosphate
major histocompatibility complex
mannose-6-phosphate receptor
4-methylumbelliferone
4-methylumbelliferyl acetate
4-methylumbelliferyl- ~-D-galactoside
4-methylumbelliferyl-cl-D-mannoside
4-methylumbelliferyl phosphate
xv
4-MU-Sia
5-NAcNeu
5-NGcNeu
NMR
NP
NP-40
PBS
PEP
PM
PNP
PS
QAE+
RBC
RER
RSB
RT
SASD
Ser
Sia
ST
Thr
UDP
VC
WGA
4-methylumbelliferyl- 0( -D-N-acetylneuraminate
N-acetylneuraminic acid (sialic acid)
N-glycolylneuraminic acid
nuclear magnetic resonance
type C Niemann-Pick disease
Nonidet P-40
phosphate-buffered saline
phosphoenolpyruvate
plasma membrane
p-nitrophenol
penicillin/streptomycin
quaternary aminoethyl
red blood cell
rough endoplasmic reticulum
reticulocyte standard buffer
room temperature
sialic acid storage disease
serine
sialic acid
sialyltransferase
threonine
uridine diphosphate
Vibrio cholerae
Wheat germ agglutinin
xvi
Chapter One
Introduction
I.A. Biochemistry of Glycoconjugates
The glycosylation of a macromolecule may contribute to and be an
intrinsic part of its physiological activity. Thus, an understanding
of the biochemical pathways leading to mature glycoconjugates is
necessary in order to appreciate fully their biological role.
I.A.l. Oligosaccharide Biosynthesis and structure
Glycosylation is a co-translational and post-translational event
occuring in the endoplasmic reticulum (ER) and the Golgi. Animal cells
contain three major classes of glycoconjugates: glycoproteins,
glycolipids, and proteoglycans. Glycoproteins carry carbohydrates
attached to serine (Ser). threonine (Thr) and/or asparagine (Asn)
residues of the polypeptide chain. Glycolipid carbohydrates are
primarily linked through the free hydroxyl on the diglyceride backbone.
Proteoglycans are composed of heteropolysaccharides in which the
terminal end is covalently attached through an a-glycosidic linkage to
a Ser residue. There may be 30-50 heavily glycosylated core proteins
1
2
in one proteoglycan molecule, and the carbohydrate may comprise more
than 50% of the total molecular weight.
Asn-linked oligosaccharides share a common inner-core structure,
as shown with a high-mannose form (Figure 1-1; reviewed by Kornfeld and
Kornfeld, 1985). This structure is synthesized enzymatically one
monosaccharide at a time on a polyisoprenoid carrier molecule called
dolichol phosphate (DolP; Figure 1-2). The term "glycose" in Figure 1-2
refers to any monosaccharide. A glycose-phosphate from a nucleoside
diphosphate is transferred to DolP to form a Dol pyrophosphate (DolPP)
glycose. Additional activated nucleotide sugars are added in a similar
fashion to the initial DolPP-glycose. The final structure consists of
DolPP linked to two N-acetylglucosamine (GlcNAc), nine mannose (Man),
and three glucose (Glc) residues (Glc3Man9GlcNAC2; as shown in Figure
1-1) •
The activated oligosaccharide is then transferred ~ bloc to a
specific Asn residue of the growing polypeptide chain, forming an N
glycosidic linkage (Figure 1-3). Only those Asn residues in the amino
acid sequence Asn-X-Thr, where X is any amino acid, can be N
glycosylated (reviewed by Hubbard and Ivatt, 1981). Not every such Asn
residue is glycosylated (Hubbard, 1987; Powell et al., 1987a), and
those that are tend to be at the N-terminal end of the polypeptide
(Pollack and Atkinson, 1983). Following transfer of the
oligosaccharide, the pyrophosphate bond of DolPP is cleaved by a
specific phosphatase to regenerate DolP.
Many additional post-translational modifications are necessary to
3
Figure 1-1. N-linked high mannose oligosaccharide. Proposed structure
of the oligosaccharide precursor in glycoprotein synthesis. Dotted
lines surround the core oligosaccharide, common to all N-linked
glycoproteins (adapted from Li et al., 1978).
N-Linked High Mannose
Oligosaccharide
Glc a1,2 t
Glc a1,3 ~
Glc 01,3 t
Man Man 41,2' a1,2 ,
Man Man 01,2 ~ a1,3",
Man
a1,2l
Man /a1,6
Man Man
01,3 ~ ~,6 Man
, ~1,4
Man
I GlcNAc
I GlcNAc
5
Figure 1-2. Synthesis of N-linked oligosaccharide. The oligosaccharide
is synthesized, one monosaccharide ("glycose") at a time, on DolP. The
final structure is shown in Figure 1-1, and the DolP is attached to the
lower GlcNAc residue (adapted from Zubay, 1983).
SYNTHESIS OF N-LINKED OLIGOSACCHARIDE
DolP Dol PP-Glycose DoIPP{Glycose)n+1
XDP - G I ycose XMP nXDP - glycose nXMP
+ nPi
7
Figure 1-3. En bloc transfer of core oligosaccharide to protein. The
oligosaccharide moiety of DolPP(Glycose)n+l (Figure 1-2) is transferred
to the amide side chain of an Asn residue on the amino acid sequence
---Asn-X-Ser/Thr---. The pyrophosphate of the released DolPP is
hydrolyzed by a specific phosphatase (adapted from Zubay, 1983).
EN-BLOC TRANSFER OF OLIGOSACCHARIDE TO PROTEIN
DoIP+ Pi
t DoIPP(Glycose}n+ I Dol PP
"""Asn-X-Ser/Thr'VV' ~ L """Asn -X-Ser/Thr"""
I (Glycose}n+1
9
convert the glycoprotein into its mature form (Figure 1-4). The three
Glc residues are rapidly trimmed from most glycoproteins while they are
still in the ER. The resulting high mannose structure can then be
modified further in the Golgi, beginning with the removal of Man
residues by o(-mannosidase. One pathway trims down to ManSGlcNAc2 and
makes limited additions such as GlcNAc and galactose (Gal). These are
referred to as hybrid structures. Hybrid structures may contain a
bisecting GlcNAc linked (1-->4) to the innermost Man residue. Another
pathway continues the previous one to build complex oligosaccharides
containing core-linked fucose (Fuc) and frequently terminated by sialic
acid (otherwise known as N-acetylneuraminic acid, S-NAcNeu). There can
be two, three or four chains emanating from the branching Man, and
these structures are therefore referred to as biantennary,
triantennary, or tetraantennary complex N-linked oligosaccharides,
respectively.
The other major structural class of oligosaccharides is a-linked
to Ser or Thr (Hanover et. al., 1982; Johnson and Spear, 1983). A
distinct set of enzymes catalyzes the synthesis of these structures.
a-linked oligosaccharides are built by monomer addition from sugar
nucleotide donors directly on the polypeptide rather than on a lipid
precursor. Trimming does not appear to playa major role. Gal and N
acetylgalactosamine (GaINAc) are typical proximal sugars, while Man is
rarely found. a-linked structures are found in both branched and
straight chain forms.
10
Figure 1-4. Post-translational modification of oligosaccharides. The
structure shown in Figure 1-1 is modified by removal of Glc residues to
yield a high mannose oligosaccharide. Removal of Man and addition of
Gal, GlcNAc, Sia and/or Fuc results in a complex oligosaccharide
(adapted from Lennarz, 1981).
Post-Translational Modification of Oligosaccharides
Glucosylated High Mannose Oligosaccharides
! a ~I"o,,"".
Man Man Man
I I I Man Man Man I '\/
Man Man
""/ Man I
GIcNAc I
GIcNAc
I """"Asn""""
High Mannose Oligosaccharide
1-Man
+ Gal, GlcNAc, Sia
+ /- Fucose
Sia Sia
I I Gal Gal
I I GIcNAc Gk::NAc I I
Man Man
~/ Man
I GIcNAc
I
Complex (biantennary) Oligosaccharide
GlcNAc - (+/- Fuc)
I """"Asn""""
12
I.A.2. Biochemistry of Sialic Acids
The sialic acids comprise a diverse family of monosaccharides
(Schauer, 1982), as seen in Figure 1-5. The abbreviations in this
figure will be used, and the generic term "sialic acid" (Sia) will be
used when no structural details are known. Numerous additional
modifications such as sulfation and phosphorylation have been reported
(Parkinnen and Finne, 1985; Lederkremer and Parodi, 1984; Freeze, 1986;
Muchmore and Varki, 1987). The acetamido methyl group can be oxidized
to hydroxymethyl, and the resulting molecule is called N
glycolylneuraminic acid (5-GcNeu; Schauer, 1982). The reason for the
diversity of sialic acids found in nature is largely unknown (Schauer,
1988) .
The anabolic and catabolic metabolism of sialic acids can be seen
in Figure 1-6 (adapted from Schauer, 1982). Sia is synthesized in the
cytosol from N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP;
Roehrig, 1984), then activated to its cytidine monophosphate (CMP)
derivative in the nucleus (Kean, 1970; Van Dijk et al., 1973; Coates et
al., 1980). The activated CMP-Sia enters the Golgi (Perez and
Hirshberg, 1986), where it is incorporated into the oligosaccharide
chain on a polypeptide by sialyltransferase (ST; Kornfeld and Kornfeld,
1985) .
The sialoglycoprotein can follow a number of pathways from this
point. The Sia moiety can be O-acetylated at carbons 4, 7, 8, and/or 9
by specific O-acetyltransferases that use acetyl coenzyme A as the
acetyl donor (Varki and Diaz, 1985). A single enzyme is thought to
13
Figure 1-5. General structure of sialic acid. Modifications of Sia
occur at carbons 4, 7, 8, and/or 9. The chemical structure at each of
these positions is indicated. Ac = acetyl.
------------- ~------.---.. - .. ----_ .... -.. -.~ .. --...
GENERAL STRUCTURE OF SIALIC ACID
OH COO-
o
R4 R7 Re Rg
S-NAcNeu H H H H
9-0Ac-S-NAcNeu H H H Ac
4-0Ac-S-NAcNeu Ac H H H
7-0Ac-S-NAcNeu H Ac H H
e-OAc-S-NAcNeu H H Ac H
e,9-diOAc-S-NAcNeu H H Ac Ac
-------------~----------~~ .
15
Figure 1-6. Metabolism of sialic acids. The pathway of Sia from the
nucleus to the lysosome is shown. Abbreviations are indicated in Figure
1-5. Enzymes are as follows:
1) CMP-neuraminate synthetase 2) Sialyltranferase (ST) 3) N-acetylneuraminate monooxygenase 4) Sialic acid 4-0-acetyltransferase 5) Sialic acid 7(8,9)-O-acetyltransferase 6) Sialic acid O-acetylesterase 7) Neuraminidase 8) N-acetylneuraminate:pyruvate lyase
(Adapted from Schauer, 1982).
HEXOSE
.1
CYTOSOL!
1 5-NAcNeu
* 5-NGcNeu
GOLGI
Metabolism of Sialic acids
* f <V I 5-NGcNeu ___ ..t_
NUCLEUS ~CTP Fpp; \~G:
CMP-5-NAcNeu 2 5-NAc Neu*
Lysosomes 5-NAcNeu PM
AcCoA ;f '-: Iii\ I ' ~ ... _,.Ac1'_~ 5
CoA /®@:'\ I ,
/ GOLGI "
pyruvate
" @/ __J~
Cytosol(?)
l , 4-0Ac-5-NAcNeu * 9-0Ac-5-NAcNeu*
* - glycoprotein acceptor
NGcMan
NAcMan
17
transfer an O-acetyl group to carbon 7. The O-acetyl group undergoes
spontaneous migration to carbon 8 and then to carbon 9, to form the
most stable ester. Addition of a second O-acetyl group results in the
8,9-diOAc-S-NAcNeu (Schauer, 1982). A different enzyme is responsible
for O-acetylation of carbon 4 (Schauer, 1982). The sialoglycoprotein
can re-enter the Golgi and undergo oxidation to the N-glycolyl
derivative (Schauer, 1982). The final destination of the glycoprotein
is the lysosome, where it undergoes partial or total degradation to its
component amino acids and monosaccharides (Conzelmann and Sandhoff,
1987). In somes cases, O-acetylation of Sia occurs prior to activation
to the CMP derivative (Higa and Paulson, 1987).
I.A.3. Protein Glycosylation: Pathway and Regulation
Protein glycosylation may be re~~lated by the pathway of movement
of the acceptor protein. N-linked glycosylation appears to occur only
in the rough endoplasmic reticulum (RER), and therefore only proteins
which possess a signal sequence are N-glycosylated (Sabatini et al.,
1982). Mannose trimming begins in the RER but can be concluded in the
Golgi. Modification of high Man forms or conversion to hybrid or
complex forms occurs in the Golgi. Nearly all glycosylation is believed
to be restricted to these two compartments. O-glycosylation appears to
be initiated and concluded in the Golgi (Hanover et al., 1982; Johnson
and Spear, 1983; Carraway and Spielman, 1986).
The glycosylation pathway as we now understand it suggests many
kinds of regulatory mechanisms. For example, each step of the pathway
18
can be influenced by enzyme synthesis and turnover. Dennis et al.
(1987) demonstrated that mutants of murine MDAY-D2 tumor cells, which
lack the enzyme GlcNAc transferase V, do not produce the tetraantennary
complex oligosaccharide structure associated with tumor metastasis.
Similarly, treatment of HL-60 promyelocytic leukemia cells with the
tumor promotor 12-0-tetradecanoylphorbol-13-acetate induces a specific
sialyltransferase, and this induction is highly correlated with cell
differentiation (Momoi et al., 1986). Another tumor promotor, orotic
acid, results in increased expression of GlcNAc transferase III
(Narasimhan et al., 1988). Elevated levels of this enzyme, which adds
the bisecting GlcNAc to complex oligosaccharides, are found in pre
neoplastic liver nodules, but are not found in normal liver cells.
Protein glycosylation may also be regulated by the supply of donor
sugar nucleotide and divalent cations. The sugar nucleotides,
synthesized in the cytoplasm or the nucleus (Kean, 1970; Van Dijk et
al., 1973, Coates et al. 1980), must be transported across the Golgi
membrane by specific transport systems (Fleischer, 1983; Perez and
Hirshberg, 1986). Variations in the translocation of sugar nucleotides
could influence the final oligosaccharide structure. The supply of
divalent cations, such as ca2+, Mg2+, and Mn2+, may also influence the
structure of the final product, as most glycosyltransferases are
dependent on one or more of these cations for enzyme activity (Watkins,
1986) .
Another possible target for the regulation of glycosylation is the
availability of the lipid cofactor DolP. Bacteria require DolP for
19
biosynthesis of cell wall peptidoglycan. The antibiotic, bacitracin,
can disrupt this process by chelating the Mn2+ ions required by the
prokaryotic DolPP phosphatase (Haavik and Froyshov, 1973).
Inactivation of this enzyme prevents regeneration of DolP, and
ultimately shuts down bacteriai cell wall biosynthesis. Chelation of
Mn2+ by bacitracin is also thought to inhibit specific
glycosyltransferases as well as the phosphatase. Another frequently
used antibiotic, tunicamycin, is a hydrophobic analogue of uridine
diphosphate GlcNAc (UDP-GlcNAc). This antibiotic blocks the addition of
GlcNAc-phosphate from UDP-GlcNAc to DolP, thereby inhibiting synthesis
of the oligosaccharide core.
The accessibility of oligosaccharide acceptors is another
important means of regulation of the glycosylation pathway. The
question of accessibility is especially important because sequentially
acting enzymes tend to be distributed vectorially through the RER and
Golgi compartments (Hirshberg and Snider, 1987). Movement of
glycoproteins through these compartments appears to be mediated by
adenosine triphosphate (ATP)-dependent dissociative movement of
vesicles (Kreisel et al., 1980). There is also an indication that some
glycosyltransferases form supramolecular complexes. Thus, it is
possible that synthesis of certain carbohydrate structures is dictated
not only by vesicle movement, but also by which enzyme cluster
initially captures the acceptor protein (Ivatt, 1985).
The incorporation of a key glycosyl residue may convert a non
substrate into a substrate for either a glycosidase or a
20
glycosyltransferase. Conversely, a substrate may be converted into a
non-substrate for the next enzyme in a given sequence, thereby
directing the oligosaccharide toward a different synthetic route
(Schachter et al., 1983). Proton nuclear magnetic resonance (NMR) of
the structures studied by Schachter et al. (1983) provides a molecular
explanation of how the regulation of the biosynthetic pathway arises
(Brisson and Carver, 1983).
I.A.4. Lectins and Their Uses
Many of the recent advances in the understanding of glycoconjugate
biochemistry have been made with the use of lectins. Lectins are cell
agglutinating proteins found in microorganisms, plants, and animals.
They bind mono- or oligosaccharides with high specificity, much as
enzymes bind substrates and antibodies bind antigens. Binding may
involve several forces including hydrophobic interactions, van der
Waals forces, and hydrogen bonding (Ochoa, 1981; Quiocho and Vyas,
1984). Binding is frequently inhibited by specific sugars in a
competitive manner.
Many lectins bind preferentially to a very limited number of sugar
structures. For example, soybean agglutinin is specific for GalNAc (Lis
et al., 1970), and to a lesser extent Gal (Hammerstrom et al., 1977).
Wheat germ agglutinin (WGA) is a cytotoxic lectin which recognizes Sia
and GlcNAc (Bhavanadan and Katlic, 1979). Peanut agglutinin is specific
for the disaccharide Gal (1-->3) GalNAc (Lotan et al., 1975) and for
Gal (Pereira, 1976). Leukophytohemagglutinin (L-PHA) is specific for
21
tri- and tetraantennary complex oligosaccharides (Cummings and
Kornfeld, 1982a).
The diversity in lectin specificity, combined with the commercial
availability and ease of purification of lectins, has made them an
important tool in biological research. One widely used application of
lectins is in the purification of macromolecules (reviewed by Lotan and
Nicolson, 1979). Lectins cross-linked to a variety of different
chromatography resins have been used in affinity purification of
glycosyltransferases (Paulson et al., 1977), histocompatibility
antigens (Bisati et ~, 1981), lysosomal enzymes (Verheijen et al.,
1981) and oligosaccharides (Cummings and Kornfeld, 1982b). The
heterogeneity of various glycoproteins has been studied with lectin
affinity immunoelectrophoresis (Breborwics and Mackiewicz, 1981).
Purification of epidermal growth factor (EGF) receptor on WGA-sepharose
is an important step in understanding the role of EGF in oncogenic
transformation (Nexo and Hollenberg, 1981). Currently, lectin-affinity
high-performance liquid chromatography is being developed for the
separation of developmentally-regulated glycoproteins that differ in
only a few oligosaccharides (Baenziger, J., personal communication,
1989).
Medicine has also benefited from the study of lectins. Many
widely used diagnostic tests are based on the interaction of tissue
samples with lectins. For example, a panel of eleven lectins was used
to classify Hodgkin's lymphoma cells, which differ in their resistance
to chemotherapeutic drugs and to radiation (Uhlenbruk et al., 1986). L-
22
PHA has been used to identify tumor cells with metastatic phenotypes
(Santer et al., 1984; Dennis et ~,1987). Lectins are currently
being used in the study of bronchial mucins from cystic fibrosis
patients, in order to identify any structural differences from normal
mucins (Van Halbeek, 1989).
A third important application of lectins in the laboratory is the
selection of mutants with altered cell surface carbohydrates. Many
lectins are cytotoxic for cultured cells, yet very little is known
about their mechanism(s) of cytotoxicity. Cells which survive this
selection protocol are deficient or chemically modified in the sugar(s)
for which the lectin is specific (Stanley, 1987). This technique has
been utilized by many researchers, and in many cases the defects have
been well-characterized (reviewed by Stanley, 1984). The Chinese
hamster ovary (CHO) cell line has been characterized extensively by
Stanley and her co-workers. Others have selected carbohydrate-deficient
clones from tumor cell populations (Finne et al., 1980; Reading et al.,
1980; Gilmer et al., 1984.) Lectin selection can also be used to select
non-metastatic variants of the highly metastatic tumor line MDAY-D2
(Dennis, 1986).
Many researchers have emphasized the need to understand the
molecular interactions between lectins and sugars, in order to use them
as probes of oligosaccharide structure-function relationships.
Considerable work on the lectin, concanavalin A (Con A) has provided
details on its subunit composition and structure (Hardman, 1979).
Studies with deoxy, O-methyl, and halogenated sugars have yielded
23
information on the topography of the binding site of Con A and the
nature of its interaction with sugars (Poretz and Goldstein, 1970;
Alter and Magnuson, 1974). Additional evidence from NMR studies (Brewer
and Brown, 1979; Bhattacharyya and Brewer, 1989) has provided more
details on its mode of oligosaccharide binding.
Similar to Con A, detailed studies of WGA and its interaction with
sugars have provided insight into previously unexplainable biochemical
and cytochemical data. Maget-Dana et al. (1981) studied the specificity
of WGA for several derivatives of ~3 ganglioside, containing altered
Sia residues, in vesicle form. They demonstrated that the Sia side
chain (carbons 7, 8, and 9 in Figure 1-5) impairs the binding of WGA to
the sugar, as its removal greatly enhances WGA binding. The carboxyl
group (carbon 1) is not involved in binding, as amidation of this group
has no effect on WGA-induced aggregation of ~3-containing vesicles.
The acetamido group (carbon 4), in contrast, is critical for binding,
as the N-glycolyl derivative has a very low affinity for WGA. Finally,
they showed that the binding of any of the sialic acids could be
reversed by GlcNAc, suggesting that the two sugars compete for the same
binding site.
X-ray crystallography and proton NMR have provided physical
evidence to support the biochemical data of Maget-Dana et al. (1981).
Wright (1984) used a high-resolution (1.8 A) electron density map to
identify two unique sugar binding sites at the subunit-subunit
interface of the WGA dimer. She proposed that small differences in
sugar binding affinities between these two sites may result from
24
different numbers of van der Waals interactions. Kronis and Carver
(1982) demonstrated that two isolectins of WGA (WGA I and WGA II) bind
5-NAcNeu linked 0(2-->3) to N-acetyllactosamine (LacNAc) with higher
affinity than the 0«2-->6) isomer. Additional studies by Kronis and
Carver (1985a and 1985b) showed that the WGA dimer has four binding
sites in solution, and each of these sites can bind either GlcNAc or 5-
NAcNeu. A tyrosine residue in the binding site is specific for the N
acetyl group of both sugars. However, the orientation of the terminal
~-G1CNAC or o(-5-NAcNeu relative to the protein surface is different,
and may lead to differential accessibility for large oligosaccharides
or membrane-bound molecules.
The mechanism of WGA binding may have important implications in
the use of WGA to select cells with altered cell-surface
oligosaccharides. structural studies on WGA-resistant B16 mouse
melanoma cells by Finne et al. (1980) showed that mutant cells had
reduced levels of 5-NAcNeu residues linked ~(2-->3) to the penultimate
Gal residues. Subsequent studies (Finne et al., 1982) indicated that
this is due to increased fucosylation of the innermost GlcNAc residue.
On the assumption that WGA resistance results from decreased binding,
the decrease in WGA cytotoxicity may be attributed to a specific loss
in 5-NAcNeu v\(2-->3) Gal sequences at the terminal position of complex
oligosaccharides.
Modification of Sia, rather than a different linkage to Gal, may
allow some cells to escape agglutination. Dennis (1986) identified
WGA-resistant MDAY-D2 mutants that expresses high levels of 5-GcNeu.
25
This is consistent with the results of Maget-Dana et al. (1981), who
showed that WGA binds poorly to 5-GcNeu. One common modification of Sia
not studied by Maget-Dana et al. (1981) is O-acetylation of carbons 7,
8, and/or 9. Preliminary evidence (Tinsley and Gilmer, 1987) suggests
that one possible explanation for WGA resistance in the WB6 variant
cell line is 9-0-acetylation of the Sia side chain (to be discussed
fully in section I.B.4.)
I.B. Functions of Glycoprotein Carbohydrates
The significance of glycoprotein carbohydrates is seen in the ABO
blood group system, first described by Landsteiner in 1900 (reviewed by
Ginsberg, 1972). Researchers have established the structures of the
antigens and have elucidated the genetic basis for the ABO blood
groups. The antigens are found on glycophorin, an integral erythrocyte
membrane protein. Individuals of blood type A have a GalNAc residue at
the non-reducing end of glycophorin oligosaccharides. Type B
individuals substitute a Gal residue for the GalNAc, and type 0
individuals have neither the Gal or the GalNAc. Type AB individuals
are heterozygous and contain both A and B structures. The gene that
determines blood type has three alleles, each of which encodes a
glycosyltranferase. The allele for type A encodes a GalNAc transferase,
type B encodes a Gal transferase, and type 0 encodes an inactive
enzyme.
As technology has improved the methods for studying glycoproteins,
26
interest in these molecules has increased. The nearly ubiquitous
glycosylation of cell surface proteins and many intracellular proteins
in eukaryotic cells has led to many studies designed to elucidate the
functional role(s) of the carbohydrates (reviewed by West, 1986).
These functions can be separated into two broad categories. Some
glycoprotein carbohydrates have specific intramolecular functions, such
as maintenance of protein conformation, proper localization, and
protection from proteolytic degradation. Others have more generalized
intermolecular functions, such as recognition phenomena, cell adhesion
and cell migration. These two interdependent roles for carbohydrates,
coupled with the fact that the carbohydrates exist mainly in
association with protein or lipid, mean that defining functional roles
for carbohydrates is difficult.
I.B.1. Non-specific Functions of Carbohydrates
Several lines of evidence suggest a generalized, non-specific
role for oligosaccharides. Many years ago, it was discovered that
carbohydrates enhanced protein solubility and protected against heat
denaturation of the protein (Schmid, 1953; Spiro, 1960). It was also
realized that oligosaccharides provided resistance to proteolytic
degradation (Gottschalk and de st. Groth, 1960), presumably because of
steric hindrance. This idea has been reinforced in numerous additional
studies (for example, Bernard et al., 1983). Recently, Kingsley et al.
(1986) identified several CHO mutants defective in glycosylation of low
density lipoprotein (LDL) receptors. The mutants exhibit low levels of
27
LOL internalization, and it is thought that the carbohydrates found on
wild-type L01 receptors may help to stabilize the molecule during
multiple rounds of endocytosis.
Oligosaccharides may protect some glycoproteins, and their removal
is frequently a signal for cell destruction. Neuraminidase treatment
of erythrocytes (Gattegno et al., 1974) and lymphocytes (Woodruff and
Gesner, 1969) showed that enzymatic cleavage of terminal Sia residues
greatly reduced the half-life of these cells in the circulation.
Removal of Sia exposes the penultimate Gal on complex oligosaccharides.
Muller et al. (1981) demonstrated that recognition of Gal, by
galactosyl-specific lectins in the circulation, may be responsible for
the sequestration and lysis of these cells.
There are also several instances where carbohydrates are known to
interfere sterically with protein-protein interaction not involving
proteolysis. Bayna et al. (1986) studied the binding of cell-surface
galactosyltransferase to GlcNAc residues on its specific
lactosaminoglycan substrate located on an adjacent cell surface. In
this system, binding of ~-lactalbumin to galactosyltransferase induces
a conformational change in the enzyme binding site so that Glc, rather
than GlcNAc, is recognized (Figure 1-7). Carbohydrate-dependent
conformational changes were also studied by Pletcher et al. (1980).
Oeglycosylation of prothrombin fragment 1 alters the degree of protein
self-association. Rapid, calcium-dependent self-association is
characteristic of normal and deglycosylated prothrombin. However, the
deglycosylated protein undergoes a secondary self-association. The
28
Figure 1-7. Alteration of galactosyl transferase specificity by o{
lactalbumin. Binding of c(-lactalbumin to cell-surface
galactosyltransferase alters the enzyme conformation such that the
active site binds to Glc rather than to GlcNAc (adapted from Bayna et
al., 1986). Abbreviations: GaIT-ase, galactosyltransferase;o{-LA,
o(-lactalbumin; LAG, lactosaminoglycan.
A Iteration of Galactosyl Transferase Specificity by a-Lactalbumin
GlcNAc
LAG
~IC-
NAc LAG
30
initial conformational change is thought to expose a site on the
protein required for additional self-association, and the carbohydrates
apparently mask this site in the native molecule.
The notion of a non-specific role for carbohydrates has received
strong support from studies on cells whose glycosylation processes have
been altered by mutation or drugs. The most dramatic studies have
involved mutant cells selected on the basis of resistance to the toxic
effects of certain lectins. Many mutations leading to loss of lectin
recognition have been identified in CHO and other cells (Stanley,
1984). Some have been characterized, and in fact most steps in the
pathway to complex, N-linked glycoconjugate formation are mutable. A
striking finding is that so many mutant cells of this type are viable
in culture.
A large body of research has focused on the role of carbohydrates
in secretory and regulatory processes. Although there are examples of
specific effects, there are considerably more examples where no
specific role for the carbohydrates could be found (Struck et al.,
1978; Sidman, 1981; Whitsett et al., 1985). In some cases, detailed
kinetic analysis has found only small effects on secretion rates (Yeo
et al., 1985). In contrast, carbohydrates may play an important role in
the regulation of eukaryotic transcription. Hart et al. (1988)
demonstrated that glycoproteins are present in the nucleus, and one
highly glycosylated nuclear protein was shown to be the RNA polymerase
transcription factor, Sp1 (Jackson and Tijan, 1988). In the latter
work, the authors showed that Sp1 bears multiple O-linked GlcNAc
31
monosaccharide residues, and suggested many possible funcional roles
for the carbohydrates. At present, most of these are non-specific and
include modulation of the half-life and stability of Sp1 in the cell,
and masking of potential phosphorylation sites on Ser or Thr residues.
These possibilities are equally plausible, as many precedents exist for
each (see above), and additional studies are underway to determine the
precise role of the a-linked GlcNAc (Hart, G. W., personal
communication, 1989).
These results suggest that carbohydrates cannot underlie essential
housekeeping functions in cells. Therefore, carbohydrates may be more
important for particular differentiated functions, which are not
expressed in cell culture systems such as the CHO cell line. This
possibility has been investigated in the Oictyostelium discoidium
system. Carbohydrate variants were isolated in which mutations have
eliminated two developmentally regulated glycoantigens (Knecht et al.,
1984; Loomis et al., 1985). Nevertheless, the cells develop normally
to produce fruiting bodies and viable spores.
other Oictyostelium discoidium variants (Gonzales-Yanes et al.,
1989) contain a modified glycoantigen which lacks the characteristic
Fuc residue. These cells may be defective in one or more enzymes that
convert guanosine diphosphate mannose (GOP-Man) to GOP-Fuc, as they are
properly fucosylated when grown in 1 roM Fuc or 1 roM GOP-Fuc. Similar
to earlier studies, this defect appeared to have little effect on cell
growth and development, although the viability of mutant spores was
somewhat lower than in the parental line (West, C. M., personal
32
communication, 1989). These results may imply that carbohydrates are
non-specific, that they serve subtle modulatory roles, or that their
role is redundant with alternate mechanisms. Alternatively, the value
of these carbohydrate structures may only become evident under the
competitive conditions of natural survival outside the laboratory.
I.B.2. Specific Functions of Glycoprotein Carbohydrates
Several examples of specific roles for glycoprotein
oligosaccharides have been demonstrated in systems other than
transformed cell lines such as CHO. One of the most thoroughly studied
systems is the mannose-6-phosphate receptor (MPR) in normal rat kidney
cells. This molecule has been identified as an essential component of a
system that in many cells allows for specific transport of newly
synthesized enzymes to lysosomes (reviewed by von Figura and Hasilik,
1986). The lysosomal recognition marker mannose-6-phosphate (M6P) is
synthesized in a two-step process (Lang ~ al., 1984). First, GlcNAc-1-
phosphate is transferred to selected Man residues on enzymes destined
for the lysosomes. The GlcNAc is then hydrolyzed, leaving M6P attached
to the glycoprotein.
The first step in lysosomal targeting is catalyzed by UDP
GlcNAc:lysosomal enzyme N-acetylglucosaminyl phosphotransferase
(Reitman and Kornfeld, 1981). This enzyme, located in the Golgi, is
selective for lysosomal enzymes (Waheed et al., 1982), and the primary
recognition determinant is on the polypeptide portion of the lysosomal
enzyme, rather than on the mannose-containing oligosaccharide (Lang et
33
al., 1984). Once targeted for lysosomes, the M6P-containing enzymes are
emptied into specialized endosomes for packaging into lysosomes
(Griffiths et al., 1988). The final step in this process is the
recycling of the MPR to the Golgi cisternae (Brown et al., 1986).
Several additional cases of carbohydrate involvement in specific
recognition are known. For example, transport phenomena may involve
specific recognition, as Hanover et al. (1989) showed that masking of
nuclear pore a-linked GlcNAc monosaccharides by WGA prevents import of
nuclear proteins. Many cell-cell interactions are also dependent on the
presence of individual monosaccharides. Bleil and Wassarman (1988)
demonstrated the requirement for terminal Gal on the oligosaccharides
of mouse egg cell sperm receptor ZP3, because oxidation of this Gal or
substitution by GalNAc reversibly inhibits sperm binding. Similarly,
the function of neural cell adhesion molecule appears to be regulated
by a specific 5-NAcNeu residue on complex oligosaccharides of this
glycoprotein (Rutishauer et al., 1985).
I.B.3. Carbohydrates and the Immune System
Carbohydrates have long been known as immunologically important
molecules (reviewed by Wu, 1988), and they may have specific and non
specific functions. An appreciation of their significance requires a
general understanding of immunology. The immune system is an
organism's defense against a wide variety of pathogens. The defense
reactions can be grouped into two major categories, based on the nature
of the effector mechanism. Reactions mediated by soluble products in
----- ------- -------
34
the body are referred to as the humoral branch, and those mediated
directly by cells are referred to as cellular branch of the immune
system (Klein, 1982). A group of molecules called lymphokines regulates
the interactions between the two branches of the immune system.
The humoral branch of the immune system consists of two main
classes of proteins: antibodies (Ab) and complement. Antibodies are
proteins that are secreted by B lymphocytes (B cells) in response to
foreign molecules called antigens (Ag). Ab belong to a special group
of serum proteins called immunoglobulins (Ig). There are five classes
of Ig molecules: IgA, IgD, IgE, IgG, and IgM. Each of these has a
unique structure and physiological role (Wall and Kuehl, 1983). The Ig
molecules consist of two heavy and two light polypeptide chains linked
by interchain and intrachain disulfide bonds. The molecules are often
Y-shaped, with Ag binding sites at the ends of each arm of the IIyII. The
portion of the stem of the Y farthest from the Ag binding sites is
called the Fc region, and is known to bind complement.
The specific binding of Ab to Ag activates a cascade of proteins
known as complement (e). The e proteins exert their effects primarily
on cell membranes, causing lysis of some cells and functional
abberations in others. Of the many effects mediated by e proteins,
lysis of red blood cells (RBe) is especially easy to measure in vitro
and has been widely adopted as a model for analyzing e and its reaction
mechanisms. The proteins themselves are diverse, high molecular weight
glycoproteins that vary greatly in physical properties, number of
chains per molecule, and biochemical function (reviewed by Joiner et
35
aL, 1984).
The cellular branch of the immune system is composed of T
lymphocytes (T cells), macrophages, granulocytes, and natural killer
cells (Golub, 1981). Broadly speaking, there are two kinds of T cells:
effectors and regulators. The effectors cause various cell-mediated
immune reactions. Delayed-type hypersensitivity (DTH) is caused by
TDTH cells. Cytotoxic T lymphocytes (CTL) cause lysis of specific
target cells. In contrast, regulator T cells control the maturation of
effector T cells and B cells. T helpers enhance, and T suppressors
block, the development of effector cells.
The other immune cell types are an equally important component of
the organism's defense system. Macrophages bind, ingest and degrade Ag,
and are thought to process and present it to lymphocytes. Unlike Band
T cells, which are Ag-specific, macrophages seem to be able to bind any
Ag (reviewed by Adams and Hamilton, 1984). Granulocytes are a type of
effector cell which acts on bacteria and parasites. These cells are
densely packed with granules containing many enzymes required for
intracellular digestion (reviewed by Gleich and Loegering, 1984).
Natural killer cells are an important defense against cancer cells that
arise in the body (reviewed by Herberman et al., 1986).
One important facet of the immune system is the cooperation
between cell types (reviewed by Singer and Hodes, 1983). This
cooperation is regulated by a set of genetically-controlled cell
surface Ag called the major histocompatibility complex (MHC; reviewed
by Dorf, 1981). This complex is known as ~ in mice and HLA in
36
humans. The MHC complex is multi-allelic, and the set of alleles
expressed by an individual is known as its haplotype. In mice, the
haplotype is designated by a superscript letter (i. e. H-2b ). The MHC
antigens are the body's way of distinguishing self from non-self.
Recognition of Ag and the appropriate self-MHC molecule, a phenomenon
known as MHC restriction, is required for cooperation between cells of
the immune system. In addition to Band T cell interactions, these Ag
playa role in transplantation (reviewed by Clift and Storb, 1987),
complement activation (Alper, 1981), and tumor immunity (Greene, 1981).
Detailed analysis of the ~ complex has been possible using
recombinant strains of mice. These animals can be divided into three
categories. Syngeneic mice are genetically identical at all loci.
Transplantation of cells or organs between syngeneic mice does not
cause an immune response. Congenic mice have different H-2 haplotypes,
but have identical genes at all other loci. These animals can be used
to study differences in the immune response, based on haplotype
differences, or they can be used to raise CTL directed only against the
H-2 molecules. Allogeneic mice are genetically different in both the
MHC and other loci. Mice immunized with cells from allogeneic strains
are called allogeneically primed. The immediate immune response in
these mice is to the foreign MHC molecules rather than to other cell
surface Ag. Another type of genetic relationship, xenogeneic, involves
individuals from different species. The immune response to xenogeneic
transplants is directed against any protein which is not highly
conserved between the species, including MHC antigens.
37
Carbohydrates have been implicated in a wide variety of humoral
immune functions. 19 secretion from B cells may be oligosaccharide
dependent (Hickman and Kornfeld, 1978). C fixation, opsonic activity,
and 19G binding to Fc receptors may require carbohydrate (Williams et
al., 1973; Koide et al., 1977). Nose and Wigzell (1983) demonstrated
that monoclonal antibodies (mcAb) from hybridomas grown in the presence
of tunicamycin, an inhibitor of N-glycosylation, have altered
immunological functions. Although they have normal protein A -binding
capacity and affinity for Ag, the mcAb cannot function in antibody
dependent cellular cytotoxicity, cannot interact with macrophage Fc
receptors, and are unable to activate C.
Glycosylation may be involved in cellular immunity, particularly
in MHC-restricted reactions. The T-cell response to Ag involves a
series of lyrnphokine-mediated events which culminate in T cell
proliferation. Ag binding to its specific receptor, in the presence of
macrophage-derived interleukin 1 (1L-1) triggers the production of
interleukin 2 (1L-2) and the generation of cell surface 1L-2 receptors
(Smith et al., 1980). Recently, Sherblom et al. (1989) demonstrated
that 1L-2 is a lectin with specificity for high Man glycopeptides, and
that the carbohydrate binding site of 1L-2 is distinct from the cell
surface receptor-binding site. Many differentiation Ag, some containing
carbohydrates, are transiently expressed and tolerated without any
apparent immune response. This may be due to cross-reactivity between
the protein determinants of self Ag and self MHC Ag (Adams, 1987).
Since not all differentiation Ag are encoded within the MHC,
38
alternative mechanisms are necessary for the induction of tolerance.
One theory is that glycosylation of cell-surface antigens is under the
influence of the MHC (Sio and Parish, 1981).
Extensive evidence exists to suggest that the MHC loci can affect
glycosylation, although the oligosaccharides on the MHC molecules
themselves are not antigenic det~rminants (Shiroishi et al., 1985).
Pimlott and Miller (1984; 1986) prepared glycopeptides by extensive
pronase digestion of plasma membrane (PM) fractions from DBA/2J P81S
mastocytoma cells. These glycopeptides inhibit allogeneic anti-P81S
CTL-mediated lysis of P81S cells in an MHC-restricted manner. The
authors suggest that the detailed structure of these carbohydrates is
under MHC control, but others have speculated that residual protein may
have served as the antigenic determinant (Powell ~ al., 1987a). Other
results provide stronger support for MHC involvement in glycosylation.
The loci controlling the activities of neuraminidase in liver (Womack
~ al., 1981) and activated T-cells (Landolfi et al., 1985) map within
the MHC. Definitive proof that oligosaccharide structure is MHC
controlled will corne with the demonstration of haplotype-specific
glycosylation patterns.
I.B.4. Functions of Sialic Acid on Glycoproteins
Many additional diverse functions are known or proposed for Sia.
Interest in these compounds has increased in recent years, as their
involvement in the biological functions of many different kinds of
molecules and cells became evident. For example, Sia plays a role in
39
cell biology by its negative charge (Jeanloz and Codington, 1976), by
influencing the conformation of glycoproteins (Aquino et al., 1980),
and by acting as receptors for viruses (Muchmore and Varki, 1987),
toxins (Helting et al., 1977), hormones (Fishman and Brady, 1976), and
Ig molecules (Itoh and Kumagi, 1980). Another important biological
role for Sia is in masking antigenic sites for a variety of Gal
specific plant and animal lectins (Schauer, 1988).
Considerable research has been done on the pathological role of
Sia. Much interest has focused on malignancies, which are often
accompanied by an elevated level of Sia in the serum (Erbil et al.,
1985). It has also been observed that many cancer cell types contain
more membrane Sia than do their normal counterparts (Alhadeff and
Holzinger, 1982). Metastasis has also been linked to sialylation of
tumor cell surfaces (Altevogt et al., 1983). The results reported by
Dennis (1986) and Schacter et al. (1983) on the relationship of
oligosaccharide branching and metastasis may be partly due to increased
sialylation of these molecules. Viral infectivity has recently been
attributed to 9-0-acetylsialic acids. Rogers et al. (1986) showed that
9-0Ac-5-NAcNeu is the preferred receptor for the hemagglutinin of
influenza virus C (IVC). The hemagglutinin contains a 9-0-
acetylesterase which functions as a receptor-destroying enzyme (Herrler
et al., 1985). Muchmore and Varki (1987) used the serine esterase
inhibitor, diisopropylfluorophosphate, to inactivate the receptor
destroying enzyme. After treatment with diisopropylfluorophosphate, IVC
binding and hemagglutination are unaffected, but the titer of viral
-------------- -------------------- ----- -----------
40
infectivity decreases by a factor of 100. The authors suggest that
inactivation of IVC receptor-destroying enzyme may be an important tool
for use in the identification of O-acetylsialic acids. The lectins
which recognize these modified Sia (Ravindranath et al., 1985) have
binding affinities too low to be experimentally useful (Varki, A.,
personal communication, 1989).
Sialic acids playa well-documented role in humoral immllnology.
For example, 5-NAcNeu is a key residue on the oligosaccharide
determinant of MN blood cell antigens (Eisen, 1980). Other work has
shown that antibodies raised against various sialoglycoproteins were
able to distinguish between positional isomers (<<-(2-->3) and 01.. (2-->6)
linkages) of Sia (Smith and Ginsburg, 1980; Hakomori et al., 1983).
Sia is also an important factor in the virulence of Streptococcus and
Neisseria meningitidis, and in the human antibody response to their
polysaccharide determinants (Jennings et al., 1984).
Sialic acids are as important in cellular immunology as they are
in humoral immunology. One way of studying the role of cell-surface Sia
is to use cells which are deficient or chemically altered in Sia.
These can be obtained in a number of ways, and the results must be
interpreted in light of the method chosen. One method is the use of
tunicamycin to inhibit N-glycosylation of newly-synthesized proteins.
Harris et ale (1981) found a slight inhibition of CTL-mediated lysis
of tunicamycin-treated target cells, but this may be due to altered
polypeptide conformation in the absence of carbohydrates. A second,
more selective method is to remove the terminal Sia with the enzyme
---------------- ---------
41
neuraminidase. The disadvantage to this is that the cells retain the
machinery to replace the Sia by either synthesizing new
sialoglycoproteins, or by resialylating cell surface proteins during
recycling. A third option is to use lectins to select cells which have
one or more defects in their oligosaccharide synthesis pathways.
All of these methods have begun to provide clues to the
immunological role of Sia. Neuraminidase-treated tumor cells have been
used as targets in cell-mediated cytotoxicity assays to try to
understand the role of Sia in immunological recognition. Gilmer et al.
(1982) performed these experiments using EL4 (H-2b ) murine lymphocytic
leukemia cells and allogeneically-primed anti-EL4 CTL from BALB/c
(H-2d) mice. They found that removal of cell-surface Sia from in vivo
EL4 target cells increases the percent cytotoxicity from 4% to 18% at
an effector:target cell ratio of 2.5:1. This suggests a masking effect
of the Sia as its removal enhanced CTL recognition and lysis of the
tumor cells.
Additional evidence in support of these results came from Powell
et al. (1987b). They found that AKTB-lb B-lymphoma cells are poor
targets for allogeneically-primed anti-AKTB CTL. In contrast, cells
treated with Clostridium perfringens (CP) neuraminidase, which
recognizes Sia alpha-linked in the 2-->3, 2-->6, and 2-->8 positions,
are 24 times better targets than untreated cells in a CMC assay.
Vibrio cholerae (VC) neuraminidase, which has the same specificity as
CP neuraminidase, yields only a six-fold increase in AKTB cell lysis.
Interestingly, CP neuraminidase releases only 75% of the Sia released
42
by VC neuraminidase. In addition, resialylation of cP-treated cells
with CMP-Sia and ST does not reduce the stimulatory capacity of the
.cells. Resialylation of VC-treated cells by addition of CMP-Sia alone
results in a 49% reversal of their stimulatory capacity, indicating
that an endogenous ST is able to replace the Sia removed by VC
neuraminidase in a selective manner. Additional exogenous ST has no
effect on the reversal of CTL lysis.
The results reported by Gilmer et al. (1982) were complicated by
the fact that the EL4 cells can continually replace the enzyme-released
Sia. To circumvent this problem, WGA was used to select clones which
are naturally low in cell-surface Sia, or which have altered Sia
structures, from the EL4 tumor cell population (Gilmer et al., 1984).
The in vivo growth characteristics and cell-surface Sia levels of the
WGA-resistant lines have been investigated in this laboratory (Table 1-
1; Tinsley and Gilmer, 1987). When the EL4 parental cell line is
injected intraperitoneally, it grows prolifically and kills the animal
within two weeks. Two of the variant lines studied, WB2 and WB4, grow
much more slowly than EL4 in ~.
One variant line, WBG, does not grow well in ~ (Table 1-1 and
Figure 1-8; Tinsley and Gilmer, 1987), suggesting that its altered
cell-surface carbohydrates may influence its recognition and clearance
by the animal's immune system. However, WBG is able to grow in vitro at
very high viability, even in the presence of high concentrations of WGA
(Figure 1-9; Tinsley and Gilmer, 1987). All of the variant tumor lines
tested thus far have lower levels of VC neuraminidase-releasable Sia
--_.--_ .. __ .... -----_.. - ... _. __ .-._ ..
43
Table 1-1 Characterization of WGA-Resistant Cell Lines
Tumor Line
EL4
WB2
WB4
WB6
WB7
WB8
WD1
Growth in WGA
+
+
+
+
+
+
# Tumor Cells (x 10-8 )1 Day 7 Day 10
5.1 4.3
0.83 3.0
0.13 1.5
0.13 0.065
N. D.3 N. D.
N. D. N. D.
0.0984 0.785
umol sia2 109 cells
0.62 + 0.06 0.63 + 0.07 -0.62 + 0.04
0.28 + .006 -0.26 + .003 -0.33 + 0.02 -0.24 + 0.01 -0.33 + 0.02 -0.34 + .003 -0.46 =. 0.03
1C57BL/6 female mice were injected intraperitoneally with 5x106 tumor cells. Peritoneal cells were harvested on day 7 or day 10. Viability was ~ 85% as determined by trypan blue exclusion.
2Neuraminidase-releasable cell-surface Sia (Gilmer et al., 1984; Tinsley and Gilmer, 1987, unpublished results), measured with the thiobarbituric acid assay (Warren, 1959).
3N. D., no data
4p. Gilmer, unpublished results.
5Measured on day 11; P. Gilmer, unpublished results.
44
Figure 1-8. In vivo growth of EL4 and WB6 in C57BL/6. 5 X 106 live EL4 ---
(8) or WBG (0) tumor cells were injected intraperitoneally. Cells were
harvested and counted on day 7 or day 10. A minimum of three animals
was used per cell line for each time interval.
-----~-~----~~ -- --~-~
109 __ ------------------------------~
o lIJ t; ....J 108 o CJ)
CJ) ....J ....J lIJ U
0: o ~ :::> J-lIJ > ....J
lL. 107
o 0:: lIJ ID ~ :::> z
Syngeneic tumor cell growth
---------------- -
e-EL4 O-W86
46
Figure 1-9. In vitro growth and viability of waG in WGA. waG cells at
a final concentration of 1 X 105 cells/mL were serially diluted with
WGA in DMEM/HS/PS. Viability and cell counts were done three days after
the addition of WGA. Growth and viability results are an average of two
cell counts per WGA concentration. There were no live EL4 cells in the
presence of WGA after three days' growth. Viability of waG was
determined by trypan blue exclusion. Symbols: 0-----0, # live cells;
o - - - 0, % viability.
>~ --.J -al « -> 0« z(!) «3: :I: z ~ 3: o c:r: (!)
<.0 al 3:
o o
o • I
• • • • • • • •
o • • • I
• • • • •
% VIASI LITY (0 -------0)
o a:>
o 1.O
-0-
o <;;t
~ N
: ~
I'o
o 0 N
i I ~ \ ____ / __ -<r...J..-______ --'rti
(00 "'0
1W/ Sl13:J 31\11 .:10 ~3811'JnN ( 0 0 )
48
than the parental line EL4 (Table 1-1; Tinsley and Gilmer, 1987).
Another WGA-resistant variant of EL4, called WD1, was selected by
Gilmer et al. (1984). This cell line grows to high viability in the
presence of WGA, similar to WB6. Its in ~ growth characteristics are
also similar to those of WE6 (Table 1-1). WD1 has less neuraminidase
releasable cell-surface Sia than does the parental line EL4. WD1 has
2.5-fold more H-2Kb molecules on its cell surface than does EL4, as
determined by Scatchard analysis of anti-H-2Kb mcAb binding (Gilmer et
al., 1984). Additional studies could not be performed on these cells,
because the line was inadvertantly lost prior to the start of the
experiments described in chapters two and three.
In light of evidence that Sia might be important in tumor cell
recognition and lysis, an investigation of the biochemical events
responsible for altered cell surface Sia in the WB6 variant line was
undertaken. WB6 was selected for initial experiments because it is the
single variant in the WE series which is unable to grow at all in the
syngeneic mouse.
The complexity of Sia metabolism offers the cell many sites at
which to regulate the extent of sialylation and Sia modification. A
reduced level of cell surface Sia may be the result of one or more
differences in enzyme activities. These will be discussed in numerical
order, as shown in Figure 1-6. Decreases in the activity of CMP-Sia
synthetase (enzyme 1 in Figure 1-6) and/or ST (enzyme 2 in Figure 1-6)
could result in lower cell surface Sia in WB6 cells. Similarly, an
increase in N-acetylneuraminate monooxygenase (enzyme 3 in Figure 1-6)
49
would convert a greater fraction of the Sia to 5-NGcNeu.
Alternatively, increases in either of the two O-acetyltransferases
(enzymes 4 and 5 in Figure 1-6) or a decrease Sia O-acetylesterase
(enzyme 6 in Figure 1-6) would produce higher levels of O-acetylsialic
acids in the variant line. These O-acetylated molecules are less
susceptible to neuraminidase cleavage (Schauer, 1982), and they have
lower extinction coefficients in the thiobarbituric acid assay by which
they are measured (Warren, 1959). The result would be an
underestimation of the total cell-surface Sia in the variant line.
Preliminary gas chromatographic data of PM isolated from EL4 and WB6 in
this laboratory suggest that WB6 has increased levels of 9-0Ac-5-NAcNeu
and decreased levels of 5-NAcNeu (Table 1-2; Tinsley and Gilmer, 1987).
These results probably represent the lower limit of the actual 9-0Ac-5-
NAcNeu levels in EL4 and WB6, as the O-acetyl group is extremely
labile. Varki and Diaz (1984) showed that most methods used to release
and purify sialic acids from glycoproteins result in extensive
destruction of the O-acetyl esters.
A third possible explanation for lower cell-surface Sia in the
variant lines is an increase in endogenous lysosomal neuraminidase
activity (enzyme 7 in Figure 1-6). Lysosomal neuraminidase was expected
to be a key point of difference in EL4 and WB6 for three reasons. The
simplest of these is that higher levels of this enzyme in the variant
cells would account for lower levels of cell-surface Sia. A second
reason is that EL4 and WB6 are tumor cells, and altered lysosomal
neuraminidase activity has been demonstrated in a number of tumor lines
50
Table 1-2. 9-0-Acetylation of Sialic Acids from EL4 and WBG Plasma Membranes1
Expt.
1
2
nmol 5-NAcNeu or 9-0Ac-5-NAcNeu/109 cell-equivalents
5-NAcNeu
50.S (51.5%)
53.4 (51.4%)
EL4 WB6
9-0Ac-5-NAcNeu
47.S (4S.5%)
50.4 (4S.6%)
5-NAcNeu
36.2 (42.S%)
31.8 (35.1%)
9-0Ac-5-NAcNeu
4S.4 (57.2%)
5S.9 (64.9%)
lSialic acids were hydrolyzed from glycoproteins with 2.0 M acetic acid for 4 hr. at SOOC. The release sialic acids were purified by ion exchange chromatography, decationized, and derivatized with trimethylsilylimidazole. Gas chromatography was performed on a column packed with 3% OV-17 on Supelcort and programmed at 200-250oC at SOC/min. 109 cell-equivalents is the amount of PM isolated from 109 cells. PM yield was 25-30%.
---------.. _-_ ... _.
51
(Miyagi et al., 1984; Yogeeswaran and Salk, 1981).
A final reason for expecting increased neuraminidase activity in
WB6 is that the parental cell line EL4 is a T cell leukemia. The MHC
antigens of T cells, but not of other lymphoid or non-lymphoid cells,
are spontaneously internalized for recycling (Tse and Pernis, 1984;
Pernis, 1985). Others have proposed that this reprocessing is selective
for the protein as well as for the cell type (Reichner et al., 1988),
and may involve passage through a lysosomal compartment en route to the
Golgi (Fishman and Cook, 1986).
I.C. Lysosomal Processing of G1ycoproteins
I.C.1. Biology of Lysosomes
The study of neuraminidase in EL4 and WB6 evolved into a broad
inquiry into the lysosomal metabolism in these cells. The lysosome is
the final destination for many cellular macromolecules. The majority of
enzymes responsible for glycoconjugate degradation in eukaryotic cells
are localized in the lysosomes. These enzymes share a number of
properties, including synthesis in the RER (Erickson and Blobel, 1979),
co-translational glycosylation (Bergman and Kuehl, 1978), and
carbohydrate modification in the Golgi (reviewed by Hubbard and Ivatt,
1981). This modification includes phosphorylation of specific Man
residues, to target the enzymes to the lysosomes (von Figura and
Hasilik, 1986). All lysosomal enzymes studied, to date, are synthesized
as precursors with higher molecular weight and are converted to the
------_.---_. -------- _ .... __ ._--_._ ..
52
mature form inside the lysosome (Holtzman, 1989). Inside the lysosome,
an acidic pH of 4.5 -5.0 (Ohkuma et al., 1982) is maintained by ATP- or
guanosine triphosphate (GTP)-dependent proton pumps associated with the
lysosomal membrane (Schneider, 1981). Correspondingly, lysosomal
hydro lases are optimally active at acidic pH values.
I.C.2. Glycoprotein Degradation
The glycosidases involved in glycoconjugate degradation are
primarily exoglycosidases, removing only the monosaccharides at the
non-reducing terminus. They are highly specific with respect to sugar
recognition and anomeric linkage, and non-specific with respect to
protein moiety. Most of these enzymes can therefore be assayed with
synthetic substrates such as the glycosides of p-nitrophenol (PNP) or
4-methylumbelliferone (4-MU).
Each of these analytical techniques has its advantages and
disadvantages. The released PNP product is assayed
spectrophotometrically, and product can be quantitated using Beer's
law. However, this method is much less sensitive than fluorescent
assays, as it is dependent on the extinction coefficient of the
chromophore (Guilbault, 1973). Fluorescence can be used to quantitate
samples in nanomolar concentrations, and the range of sensitivity is
much greater than for spectrophotometric assays. The disadvantage of
fluorescence is that quantitation is indirect, and depends on
fluorescence intensity of a sample relative to a set of standards.
Degradation of complex carbohydrates requires the sequential
53
action of various glycosidases, sulfatases, phosphatases, and esterases
(Conzelmann and Sandhoff, 1987). The deficiency of one such lysosomal
enzyme can therefore block the catabolism of several glycoconjugates.
The non-degradable material then accumulates in the lysosomes, giving
rise to one of several lysosomal storage diseases. For example,
neuraminidase (sialidase) deficiency causes the accumulation of
sialyloligosaccharides in sialidosis patients. Multiple lysosomal
enzyme defects in I-cell disease result in glycolipid and glycopeptide
accumulation. ~1 gangliosidosis is caused by defective lysosomal ~
galactosidase, and results in storage of asialo-~l ganglioside and
galacto-oligosaccharides (Durand and O'Brien, 1982).
The precise sequence of glycoprotein catabolism is not yet known.
The protein core is degraded by several lysosomal proteases. The
accumulation of glycopeptides rather than intact glycoproteins in many
storage diseases indicates that the proteases can hydrolyze the
polypeptide before the oligosaccharides are removed (Durand and
O'Brien, 1982). The heterogeneity of the carbohydrate portion of
lysosomal enzymes isolated from normal tissues, in contrast, indicates
that deglycosylation normally precedes or is concurrent with
proteolysis (Willcox and Renwick, 1977; Swallow et al., 1984). The
same conclusion can be drawn from experiments demonstrating a longer
half-life of the protein core than of the oligosaccharide chains in
membrane glycoproteins (Kreisel et al., 1984).
--------------------------- ------- ---------
54
I.C.3. Defects in Lysosomal Transport
While the lysosomal catabolic functions have been widely
recognized for decades, the fate of the small molecules produced by
acid hydrolysis has not been well studied. However, certain newly
discovered metabolic disorders have revealed the existence of a process
of carrier-mediated transport of sugars and amino acids across the
lysosomal membrane. The consequence of a mutation in lysosomal
transport would be a storage disease, biochemically resembling other
lysosomal storage disorders, except that the stored material would be
monomeric rather than polymeric (Forster and Lloyd, 1988).
Currently, three disorders are known to result from impaired
lysosomal transport. These diseases have a wide range of clinical
symptoms and a wide variation of phenotypes, ranging from mild to
severe forms of each disease. Cystinosis involves the disulfide amino
acid cystine (Smith ~ al., 1987), Type C Niemann-Pick disease (NP)
involves unesterified cholesterol (Sokol et al., 1988), and sialic acid
storage disease (SASD) involves Sia (Renlund ~ al., 1986a and 1986b;
Mendla, 1988). In each case, the stored substance is free and not part
of a macromolecular structure. These three disorders serve as
prototypes for metabolic diseases of lysosomal membrane transport.
I.C.3.a. Cystinosis
The clinical pathology of cystinosis is attributed to the
lysosomal accumulation of 10 - 1000 times the normal amount of cystine,
which crystallizes in many tissues of the body, including the kidney,
-------------------------------------- --------------
55
liver, spleen, and intestine. Although the muscle was previously
considered to be spared, cystinotic muscle cells in vitro and in vivo
do store cystine (Harper ~ al., 1987). Cystinosis patients begin
exhibiting acute renal failure at age 6-18 months. The failure of the
kidneys to resorb water, glucose, potassium, phosphate, and bicarbonate
results in polyuria and dehydration, glucosuria, hypokalemia,
hypophosphatemic rickets, and acidosis, respectively. Kidney function
slowly deteriorates, so that by 10 years of age, affected children have
lost virtually all renal function. The patients then require dialysis
or renal transplantation, which has successfully prolonged the lives of
many patients with cystinosis (Gahl et al., 1986).
Cystine is generated inside lysosomes by the cathepsin-catalyzed
breakdown of proteins (Thoene et al., 1977). Cystine produced in the
lysosomes normally enters the cytoplasm, where it is reduced to
cysteine and used for the synthesis of glutathione and proteins.
Studies utilizing Epstein Barr virus-transformed human lymphoblasts
(Jonas et al., 1983), human diploid fibroblasts (Jonas et al., 1982)
and human leukocytes (Gahl et al., 1982a and 1982b) have demonstrated a
specific lysosomal transport system for cystine. Gahl and his co
workers demonstrated that lysosomal cystine tranport is a saturable
process, with a Vmax of 3 pmol of 1/2 cystine per minute per unit of
hexosaminidase activity. Patients heterozygous for cystinosis have half
the maximum velocity of lysosomal cystine egress, and homozygotes have
negligible velocities regardless of the level of cystine loading.
Although saturability of cystine egress strongly suggested
------ ------------ ---------------
56
carrier-mediated transport, proof was forthcoming in the demonstration
of counter-transport (Gahl et al., 1983 and 1984). In this process,
trace amounts of a radioactive substance will cross a membrane at an
increased rate if there is a substantial concentration of the
nonradioactive substance on the opposite side of the membrane. In these
experiments, lysosomes loaded with non-radioactive cystine take up more
(3H)-cystine than do normal lysosomes not previously loaded with
cystine. Cystinotic lysosomes take up virtually no (3H)-cystine, and
heterozygotes take up half the normal amount of cystine. This suggests
that transport of cystine to the cytoplasm is defective in these cells,
since they are unable to exchange lysosomal cold cystine for
cytoplasmic 3H-cystine. The carrier is specific for the L-isomer of
cystine, since D-cystine does not compete for (3H)-cystine uptake. In
addition, homocystine is not recognized by the carrier, nor was
o-carboxyethyl-L-thiocysteine, which is cystine without one amine
group. These data testify to the specificity of ligand binding by the
normal lysosomal cystine carrier. All of these studies have shown that
the lysosomal cystine transport system is deficient in lysosomes from
individuals with the autosomal recessive disease, cystinosis.
The biochemical defect responsible for lack of cystine transport
to the lysosome in cystinosis is currently under investigation.
Recently, studies on cystine dimethyl ester (CDME)-loaded human
diploid fibroblasts have suggested that cystine transport out of normal
lysosomes is regulated by both the lysosomal membrane potential
gradient and the transmembrane pH gradient (Smith et al., 1987). When
------_ .. _--_. __ ._. --'-"'-'"
57
lysosomes are incubated with 5 roM MgC12 and 2 roM ATP (Mg/ATP), the
amount of lysosomal cystine lost from normal fibroblasts doubles, but
the amount of cystine lost from cystinotic lysosomes remains
negligible. The effect of Mg/ATP on loss from normal fibroblast
lysosomes is abolished when either carbonyl cyanide m-chlorophenyl
hydrazone or N-methylmaleimide is present, suggesting that the effect
of Mg/ATP was mediated by the action of a lysosomal proton-
trans locating ATPase. In all of the experiments involving cystinotic
lysosomes described above, other cystine metabolizing systems appear to
be present in the mutant cells, including cystine transport in the
plasma membrane (Forster and Lloyd, 1985).
I.C.3.b. Type C Niemann-Pick Disease
Like cystinosis, NP results from defective transport of a small
molecule from the lysosomes to the cytoplasm. In NP patients,
LDL uptake by the mutant cells leads to an excessive intracellular
accumulation of unesterified cholesterol (Kruth et al., 1986). It is
believed that the disruption of normal cholesterol metabolism causes
the neurological pathologies associated with the disease (Crocker,
1961). Unlike Type A and Type B NP disease, which result from
sphingomyelinase mutations (Schneider and Kennedy, 1967), no enzyme
defect can be found in NP (Vanier et al., 1986).
Additional studies were done to rule out any differences in LDL
binding and/or endocytosis. Ordinarily, LDL and its receptor are
endocytosed and transported to acidic endosomes for the dissociation of
58
receptor and ligand. The 1D1 then enters the lysosomes for degradation
into lipid monomers and amino acids. When normal and NP fibroblasts
are incubated with 1D1 for 24 hours, total cellular cholesterol levels
increase 2.23-fold and 2.28-fold, respectively, suggesting that
cholesterol uptake is sL~ilar in the two cell lines (Sokol et al.,
1988). However, the cholesterol distribution varies greatly. Before
1D1 addition, both cells have 16%-18% of the total cholesterol in the
plasma membrane (PM). After incubation, normal cells have 46% of the
total cellular cholesterol in the PM, while in NP cells the value is
17%. The excess cholesterol in the NP fibroblasts is sequestered in
the lysosomes rather than the PM.
The relationship between lysosomal storage and deficient
intracellular mobilization of cholesterol in NP suggests several
possible disruptive mechanisms. Excessive cholesterol accumulation may
represent a primary defect in the lysosome itself. It is also possible
that lysosomal cholesterol accumulation simply reflects the capacity
and availability of lysosomes to store cholesterol when they are called
upon to do so because of some more primary defect. Potential primary
post-lysosomal abnormalities could include deficient sterol carrier
proteins or lesions in membrane interactions which normally serve to
transport cholesterol to specific targets. The heterogeneity in the
clinical presentations of Type C NP patients (Vanier et al., 1986) may
be pertinent for a fuller understanding of the clinical and molecular
pathogenesis of this disease.
------~---------- --------
59
I.C.3.c. Sialic Acid Storage Disease
Similar to NP patients, SASD patients have highly heterogeneous
clinical features. All variants of SASD are characterized by severe
psychomotor and mental retardation, coarse facial features, small
stature, and neurological degeneration (Autio-Harmainen et al., 1988;
Baumkotter et al., 1985). However, striking differences are found in
the progression of the disease and the lifespan of the patient.
Patients with Salla disease present severe symptoms and early onset,
but a slow progression of the disease (Renlund et al., 1983a). In
contrast, patients with the severe infantile form die in early
childhood (Tondeur et al., 1982).
Biochemically, all types of the disease are characterized by
accumulation of free Sia in lysosomes in several organs and in cultured
fibroblasts (Hancock et al., 1983), and by excessive urinary excretion
of free Sia (Renlund et al., 1979; Paschke ~ al., 1986a). The
biochemical defect underlying these diseases is so far unknown. All the
enzymes known to be involved in Sia metabolism were proven to be
normally active in liver and cultured fibroblasts from patients with
SASD (Renlund et al., 1983b). It was hypothesized by many researchers
that SASD resulted from defective transfer of Sia across the lysosomal
membrane into the cytosol, and much of the recent evidence supports
this idea.
Several groups of reseachers have undertaken metabolic labelling
of normal and SASD cells to determine whether SASD is a consequence of
a defect in the lysosomal transport of Sia. One method is to incubate
-------~--~----- -~~- ~----~-~-- --
60
cells with free Sia. The results of these experiments must be
interpreted with caution, as studies by Hancock et al. (1983) showed
that the negative charge on Sia at physiological pH prevents its uptake
by cultured fibroblasts. Jonas (1986) incubated normal and SASO
fibroblast lysosomes with free Sia and studied the rate of Sia loss. He
showed that after 9 minutes, normal loaded lysosomes contain only 30%
of the Sia that they contained at zero time. In contrast, SASO
lysosomes contain 90% of their initial Sia after 9 minutes, suggesting
an inability of the Sia to exit from the lysosomes. However, the
negative charge may also play a role in the uptake of free Sia by
fibroblast lysosomes, since Jonas reported the Sia content at time zero
was 5.0 nrnol/unit ~-hexosarninidase for normal fibroblasts, but was 229
nrnol/unit !3-hexosarninidase for SASO fibroblasts.
These problems were overcome by Mancini et al. (1986), who used a
tritium-labeled Sia methyl ester to study Sia metabolism in cells from
SASO patients. After incubating the cells for varying time periods,
they separated the methyl ester metabolites on thin-layer
chromatography, and counted the radioactivity corresponding to free
Sia. The half-time for clearance of (3H)-Sia could then be determined
for the normal and diseased cells. Like Jonas, they found that the
rate of Sia loss from SASO lysosomes is much less than that of normal
cells. The T1/ 2 values for control fibroblasts and infantile SASO
fibroblasts are 55 minutes and 146 minutes, respectively. Adult SASO
fibroblasts had an infinite T1/ 2 , because there was essentially no
release of free Sia from these cells. The results of these
61
experiments, where the conversion of methyl ester into free Sia is
independent of neuraminidase activity, exclude the possibility that
increased lysosomal Sia storage is due to increased neuraminidase
activity or any other mechanism of Sia over-production.
Much of the more convincing evidence for defective lysosomal Sia
transport in SASD comes from studies using ManNAc, the metabolic
precursor of Sia (Roehrig, 1984; Diaz and Varki, 1985, and Figures 1-10
and 1-11). Paschke et ale (1986b) used (3H)-ManNAc to investigate the
accumulation, distribution, and metabolic fate of labeled (3H)-Sia.
Normal and SASD cells incorporate (3H)-ManNAc into sialoglyconjugates
at the sam~ rate, but the diseased cells show a non-saturable lysosomal
accumulation of free Sia following sialoglycoconjugate degradation.
Accumulated Sia cannot be chased from SASD cells, although N-acetyl
(3H)-hexosamines (HexNAc) appear in the chase medium. Their
observation of markedly increased ratios of (3H)-Sia/(3H)-HexNAc
suggests that the Sia is less accessible to the processes of
degradation and utilization. Additional studies by Paschke et ale
(1986b) were done to determine the intracellular location of the stored
Sia. Digitonin is a steroid-oligosaccharide conjugate that
permeabilizes membranes by complexing with cholesterol (Zuurendonk and
Tager, 1974). The PM is much more sensitive to digitonin than are
organelle membranes, so titration with increasing concentrations of
digitonin is a means of identifying the site of metabolite storage. The
release of (3H)-Sia follows closely the digitonin-induced release of
the lysosomal enzyme ~-hexosaminidase, suggesting a lysosomal location
------~---------~-
62
Figure 1-10. Synthesis of N-acetylneuraminic acid. ManNAc is
phosphorylated at the hydroxyl of carbon 6, then undergoes condensation
with PEP to yield N-acetylneuraminate-9-phosphate. Hydrolysis of Pi
produces Sia (adapted from Roehrig, 1984).
Synthesis of N-acetylneuraminic Acid
N-acetylmannosami ne
HOH2C-CHOH - ~HOH
H3C\ )NH C II o
OH
o
N- acetylneuraminic Acid
ATP AOP
"=- .A ..
N-acetylmannosamine -6-phosphate
~OH2C-CHOH-~HOH
H3C, /NH ..... a--,-=_-- C II o
Pi OH
Pi
o
N-acetylneuraminate-9- phosphate
64
Figure 1-11. Metabolic pathways of N-acetylmannosamine. A eukaryotic
cell is shown, and a lysosome is represented by the oval within the
cell. Sia derived from sialoglycoconjugates is cleaved by
neuramindase, then is transported into the cytoplasm. The closed circle
on the arrow connecting lysosomal and cytoplasmic Sia represents the
proposed site of the biochemical defect in SASD (adapted from Renlund
et al., 1986b).
Metabolic Pathways of N-acetylmannosamine
ManNAc
pyruvate
ManNAc _ r~ + ~ Sialic acid
+ ~
Sialic Acid
CDl CMP- Sialic
Acid
sialoglycoconjugates
amino acids diglycerides
monosaccharides
amino acids diglycerides monosaccharides
66
for the stored material.
Studies on purified lysosomes by Renlund and his colleagues
(1986a) confirmed the lysosomal accumulation suggested by earlier
results. They incubated normal and SASO fibroblasts with varying
concentrations of non-radioactive ManNAc and followed the Sia synthesis
and lysosomal egress using HPLC. Their results indicate that in normal
fibroblasts, the velocity of Sia egress is proportional to the initial
level of loading with ManNAc. SASO fibroblasts show a negligible rate
of Sia egress, regardless of the level of ManNAc incubation. However,
they were unable to demonstrate saturation of Sia egress. This is
consistent with either a diffusional or a facilitated transport system,
since demonstration of saturation kinetics may require much higher
concentrations of ManNAc than would be encountered under physiological
conditions. They were unable to demonstrate defects in the transport of
another lyososomal degradation product, cystine, which suggests that
the defect in SASO is restricted to Sia transport and is not a
generalized lysosomal membrane defect.
Additional work by Renlund and co-workers (1986b) helped to rule
out other possible explanations for the differences in normal and SASO
fibroblasts. Using (3H)-ManNAc, Renlund found that labelling of free
Sia was 5-10 times higher in the diseased cells than in the normal
cells, and that the loss of Sia by SASO cells is significantly lower
than it is in normal cells. In order to rule out a more generalized
defect in lysosomal glycoprotein and glycolipid degradation, he and his
co-workers studied the binding, internalization, and lysosomal
67
breakdown of LDL. Because of its relatively high Sia content (Enholm
et al., 1972), it was considered to be a suitable vehicle for Sia
transport into the lysosome. They labelled LDL in its Sia moiety, using
Na periodate oxidation and NaB3H4 reduction (periodatejNaB3H4) or in
its protein moiety (125I ). Using the radioactive LDL, they demonstrated
that in SASD cells LDL-derived free Sia accumulates in the lysosomes,
whereas the LDL protein moiety is catabolized normally.
Similar experiments were done by Mendla et al. (1988), using the
highly sialylated glycoprotein fetuin. Endocytosis of fetuin is not
receptor-mediated, as is LDL endocytosis, and its use, therefore,
eliminates one potential variable in Renlund's work. Localization of
the (3H)-Sia derived from periodatejNaB3H4-labelled fetuin in normal
and SASD fibroblasts showed that, although the total amount of label is
the same in the two cell lines, most of the labeled Sia is cytosolic in
normal cells and lysosomal in SASD cells. In addition, SASD lysosomes
exhibit a marked decrease in their density, which may be due to the
influx of water following the osmotic gradient produced by the
accumulated Sia. Taken together, these findings show that fetuin
derived Sia normally enters the cytosol following lysosomal
degradation, and in SASD fibroblasts this process is disrupted by the
defective lysosomal transport system for Sia.
Taken as a whole, these results provide strong evidence for the
hypothesis that the basic defect in SASD involves the transport of free
Sia out of the lysosome. As a result, its further catabolism is
prevented because the enzyme that degrades Sia, N-acetylneuraminate
----------------------------------
68
pyruvate lyase (Figure 1-11, enzyme 4), is located in the cytosol. A
generalized defect of lysosomal glycoprotein catabolism or solute
translocation is unlikely, because LOL and fetuin protein products are
released normally, and because cystine crosses the lysosomal membrane
in SASO fibroblasts at the same rate as it does in normal fibroblasts.
SASO is a new example of an inherited lysosomal transport disorder, and
the first to involve a carbohydrate molecule. No one has been able to
identify the solute carrier in any of the lysosomal transport defects
discussed above, and the molecular nature of the carrier is still
uncertain. Considerable work remains to be done in identifying the
transport mechanisms in normal cells. study of lysosomal transport
processes may be of increasing importance in defining a whole new group
of lysosomal storage diseases.
I.O. Overview of this Research
EL4 and its WGA-resistant clone WB6 have very different growth
properties in vivo (Table 1-1) and in the presence of WGA in ~
(Figure 1-9). EL4 is omitted from Figure 1-9 because it does not grow
at all in the lowest concentration of W~A. Several immunological
approached were used in previous studies (Gilmer et al., 1982; Gilmer
et al., 1984) in an attempt to understand the in vivo growth of these
cells. The experiments described here focus on the metabolism of the
cell-surface molecules that may underlie the differences in the
immunogenicity of EL4 and WB6.
In order to test the hypothesis that lysosomal processing of cell-
--------------------
69
surface antigens may result in altered physico-chemical and growth
properties in WB6, several lysosomal hydrolase activities were studied
in EL4 and WB6. 4-MU conjugates were used as substrates to measure
acetyl esterase, acid phosphatase, ~ -galactosidase, 0<. -mannosidase,
and neuraminidase. The fate of the Sia released by neuraminidase was
studied using cold ManNAc and [6-3H]-ManNAc. The results reported here
demonstrate that EL4 and WB6 have differences in lysosomal processing
of sialoglycoconjugates, and in the transport of lysosomal Sia into the
cytoplasm.
Chapter Two
Materials and Methods
II.A. General Methods
II.A.1. Reagents and Equipment
The source{s) of each chemical used in these experiments can be
found in Table 2-1, at the end of this chapter. The equipment supplier
is listed within the text of the appropriate "Materials and Methods"
section. Abbreviations for reagents in Table 2-1 can be found in the
"List of Abbreviations" starting on page xiv.
II.A.2. Cell Lines
EL4, a murine lymphocytic leukemia line of T-cell origin derived
from CS7BL/6, is available from the Salk Institute, La Jolla, CA. WB6
is a stable variant of EL4, isolated in our laboratory by a single-step
selection in soft agar containing 3.2~g/mL WGA (Gilmer et al., 1984).
Cells were grown in 7S cm2 Corning tissue culture flasks (Fisher
Scientific, Pittsburg, PAl or in spinner flasks (Bellco Biotechnology,
Vineland, NJ) in a S% CO2 , water-jacketed incubator (Forma Scientific,
Marietta, OH) at 370 C to a density of (O.S - 1.0) X 106 cells/mL.
70
71
Cells were passed every 2-3 days in Dulbecco's Modified Eagle's Medium
(DMEM) containing 10% heat-inactivated horse serum (HS) and 1%
penicillin-streptomycin (PS). Cell viability ranged from 85% to 95%, as
determined by trypan blue exclusion.
II.A.3. Protein Assay
Samples were solubilized in 0.5% (w/v) Na taurocholate at OoC for 15
minutes. Insoluble membrane fragments were pelleted for 5 minutes at
highest speed in an Eppendorf microcentrifuge. Supernatant protein was
measured by the Folin method using taurocholate-solubilized bovine
serum albumin (BSA) as a standard (Lowry et al., 1951)
II.B. Cell Fractionation in RSB
II.B.1. Cell Disruption by Nitrogen Cavitation
Cells were disrupted according to Crumpton and Snary (1974), as
modified by Gilmer et al. (1982). Cells were centrifuged at 300 X gav
and washed twice in room temperature (RT) phosphate-buffered saline
(PBS). Cells were resuspended at 1% (v/v) in Reticulocyte Standard
Buffer (RSB; 10 roM Tris-HC1, 1.5 mM MgC12 , 0.2 mM CaC12' 10 roM NaCl, pH
7.4) at OoC. The cells were equilibrated in a nitrogen (N2) cavitation
bomb (Parr Instruments, Moline, IL) at 150 pounds per square inch for
13 minutes and were kept suspended by a magnetic stirrer. Cells were
released dropwise from the N2 bomb to maximize cell rupture, and kept
on ice until further use.
72
II.B.2. Fractionation of Cells Disrupted by N2 Cavitation
Cell fractionation was performed as shown in Figure 2-1 (Miyagi
and Tsuiki, 1984). All steps were performed at 0-4oC. Centrifugations
were done in a Sorvall RC-5B centrifuge (Du Pont Instruments, Newtown,
CT) with either SA-600 or SS-34 fixed angle rotors. The cell lysate
was centrifuged at 300 X gav to remove nuclei and unbroken cells, and
the resulting supernataut was centrifuged at 4000 X gav for 15 minutes
to pellet the lysosomes and mitochondria. The 4000 X gav pellet was
resuspended in five volumes ice-cold 0.25 M sucrose/1.0 mM EDTA and
centrifuged at 11,000 X gav for 20 minutes. This pellet was
resuspended in 0.25 M Na acetate, pH 4.6 and used as the lysosome
source in glycosidase experiments. The 4000 X gav supernatant was
centrifuged at 20,000 X gav for 30 minutes to separate the PM, ER, and
Golgi from the cytosolic components. The pellet was resuspended in 36%
(w/w) sucrose/10 mM Tris-HC1, pH 7.4, dounced 20 times on ice, and
diluted to 28 mL, using the same buffer, in an IEC centrifuge tube. 8
mL of 25% sucrose (w/w)/10 mM Tris/HC1, pH 7.4, was layered carefully
above the 36% suspension to within 5 mm of the top of the tube. The
samples were centrifuged at 90,000 X gmax (23,000 RPM in an IEC #485
swinging bucket rotor) in an IEC model B-60 ultracentrifuge at 4°C for
18 hours with the brake turned off.
The sucrose gradient was analyzed on an Auto Densi-Flow (Buchler
Instruments, Fort Lee, NJ) connected to an absorbance monitor (model
UA5, Instrumental Specialities Co., Lincoln, NE) and chart recorder
-----------~-. ----~----
73
Figure 2-1. Cell Fractionation in RSB. Cells were disrupted according
to Crumpton and Snary (1974), as modified by Gilmer et al., 1982. The
4000 x gav pellet was purified according to Miyagi and Tsuiki (1984).
Cell Pellet
t 1% (v/v) Suspension In RSB
t Nitrogen bomb
Cell Lysate I 300 X g. 15 min.
~ l pellet supernatant
I 4000 X g, 15 min. (nuclei and unbroken cells)
~ l supernatant pellet
(milo., Iyso.) 20,000 X g, 30 min ••
11,000 X g, 20 min.
pellet
("Iysosomes")
0.25 M sucrose
supernatant pellet (mlcrosomes)
Spero.. gr.dl.nl 90.000 x g. 18 hr •.
ER (pellet)
PM (Interface)
Goigi
(surface)
supernatant
(cytoplasm)
75
(Hitachi-Perkin Elmer, Tokyo). The first four 30-drop fractions from
the top of the tube contained the Golgi apparatus. The PM was at the
interface between the 25% sucrose and the 36% sucrose layers. The ER
membranes pelleted at the bottom of the tube. The fractions containing
each of these organelles were pooled and stored in sterile freezing
vials at -aooc until further use.
II.C. Assays Utilizing Fluorescent Substrates
II.C.l. Fluorescence Measurements
4-MU fluorescence was measured on a Hitachi Perkin-Elmer MPF-2A
fluorescence spectrophotometer. Excitation was at 365 rum, and emission
was at 450 rum. The instrument was supplemented with an external cooling
bath for OOC circulation in the sample chamber. The chamber was
modified for dry gas input to minimize condensation on the cuvette
walls. All experiments were performed in 1 cm cuvettes using a lamp
slit width of 6JUm. Ratio recording was used with a reference
sensitivity of 3. A standard curve of serially diluted 4-MU was used to
convert relative percent fluorescence to rumoles 4-MU in experimental
samples.
II.C.2. Enzyme Assays
Five 4-methylumbelliferyl conjugates were used to assay lysosomal
acid hydrolase activity, as shown in Figure 2-2. Enzymatic cleavage
yields ROH and 4-MU. The latter can be assayed spectrofluoro-
-----------_ ... - --_._-----
76
Figure 2-2. Enzymes Assays Utilizing 4-MU Substrates. Five 4-MU
conjugates were used as substrates in lysosomal acid hydrolase assays.
4-MU product formation was monitored spectrofluorometrically, with
excitation at 365 nm and emission at 450 nm.
Enzyme Assays Utilizing 4-Methylumbelliferone Substrates
R group
o II -o-pI
-0
HOCH2 -CH-OH I CHOH
CH3-C-NQCOOH II o
OH
----- ----------
specific hydrolase
R-OH
4- Methylumbelliferone
Aexc = 365nm
"em = 450nm
enzyme specificity
Acetyl esterase
Acid phosphatase
{3 - Galactosidase
a - Mannosidose
Neuraminidase
78
metrically, and quantitated via a standard curve. Assays were
performed in the region of linear product formation over time, so that
the amount of fluorescence is a measure of enzyme activity. Enzyme
specificity is governed by the nature of the R group, so that a single
standard curve of the 4-MU product can be used to measure the activity
of a number of different enzymes.
Enzyme assays were carried out, as described by Alhadeff and Wolfe
(1981) with several modifications. Major exceptions are noted in the
"Acetylesterase Activity" section. Reactions were performed in
duplicate or, whenever possible, triplicate. All procedures were
carried out at 00-40C unless otherwise stated. Enzyme volume (protein,
buffer, and detergent) was 0.120 mL per reaction. Heat-inactivated
controls were prepared in an identical manner, and placed in a boiling
water bath for 1 hour. Experimental and control samples were
centrifuged for 10 minutes at highest speed in an Eppendorf
microcentrifuge to pellet insoluble material. 0.120 mL aliquots of the
supernatant were transferred to 12 x 75 rom glass or plastic test tubes.
4-MU substrates were diluted from 10 roM stock solutions in methanol.
0.060 mL of the diluted solution was added to each reaction tube to
give the desired final concentration in 0.180 mL final reaction volume.
Tubes were centrifuged briefly at 300 X gav to remove any enzyme or
substrate droplets from the walls of the tube. Samples were placed in
a 370C water bath for the desired time period. Following incubation,
tubes were placed on ice, and the reactions were terminated with 3.0 mL
ice-cold 85 roM glycine-carbonate (gly-carb) buffer, pH 9.4.
79
Fluorescence and product determination were done, as described in
"Fluorescence Measurements".
II.C.2.a. Neuraminidase Activity
Reactions were set up as described above. Cell samples containing
0.150-0.200 mg protein per reaction in 0.25 M sodium acetate, pH '4.6,
were solubilized in 0.5% final concentration Triton X-l00, NP-40, or
sodium taurocholate. The substrate was 4-methylurnbelliferyl-N
acetylneuraminic acid (4-MU-Sia).
II.C.2.b. p3-Galactosidase and ~-Mannosidase Activity
Enzyme reactions were carried out, as described, for neuraminidase
activity, except that 1 roM 4-methYlurnbelliferYl-~-galactoside (4-MU
Gal) and 1 mM 4-methylurnbelliferyl-o(-mannoside (4-MU-Man) were
substituted for 4-MU-Sia. In addition, since there was insignificant
substrate hydrolysis by heat-inactivated controls or enzyme blanks,
these steps were routinely omitted.
II.C.2.c. Acid Phosphatase Activity
Enzyme reactions were conducted, as described for neuraminidase,
except that the reaction buffer was 50 roM Na citrate, pH 4.6, and the
substrate was 4-methylumbelliferyl phosphate (4-MU-P). Correction for
spontaneous substrate hydrolysis was done using heat-inactivated
controls.
80
II.C.2.d. Acetylesterase Activity
Samples and heat-inactivated controls were prepared, as described,
by Sparkes et al. (1979). Aliquots of cell homogenates (0 - 0.5 mL)
were brought to a total volume of 3.5 mL in 10 roM Na acetate, pH 5.5.
The samples were allowed to warm up to room temperature, then the
reaction was initiated by the addition of 0.05 mL 4-methylumbelliferyl
acetate (4-MU-Ac, 10 mM in methanol). The cuvette was inverted quickly
to mix, and product formation was followed in situ. Fluorescence was
recorded over the linear time period, and recording was terminated when
fluorescence exceeded 100% or when there was no further increase in
product formation. As the experiments were done at acidic pH, product
fluorescence was greatly decreased (see Chapter three). The higher
sample sensitivity thus required led to greater instrumental noise. The
resulting decrease in reproducibility necessitated 5-6 runs of a given
sample dilution, in contrast to other 4-MU assays done in duplicate or
triplicate.
11.0. Lysosomal Sensitivity to NaCl
Cells were disrupted in RSB with N2 cavitation, as described
above, except that the NaCl concentration in RSB was varied from 10 mM
to 100 mM. Cell fractionation was terminated after the 4000 X gav
centrifugation. This is the first step in Figure 2-1 in which cytoplasm
and "lysosomes" were separated, and is therefore a way to track
lysosomal rupture caused by the buffer. The 4000 X gav pellet and 4000
81
X gav supernatant were assayed for ~-galactosidase activity (as
described above) to determine the extent of lysosomal rupture.
II.E. Purification of Lysosomes in Isotonic Sucrose
Lysosomes were purified, as described by Harms et al. (1981).
The procedure is outlined in Figure 2-3. Briefly, the cells were
centrifuged at 300 x gav and washed twice in RT-PBS. All subsequent
steps were carried out at 4oC. Cells were resuspended in Lysosome
Isolation Buffer (LIB; 0.25 M sucrose, 10 roM triethanolamine, 10 roM
acetic acid, 1 roM EDTA, pH 7.4) at a density of 2 X 107 cells/mL, and
lysed with 10 strokes of a glass/teflon homogenizer (Thomas Scientific,
Philadelphia, PA). Unbroken cells and nuclei were removed by
centrifugation at 750 X gav. This procedure was repeated 3 times, and
the supernatants were combined and passed through one layer of pre
washed filter paper (7.5 cm, qualitative, American Scientific Products,
MCGaw Park, IL) in order to remove any solids dislodged from the 750 X
gav pellet. The filtrate was centrifuged for 30 minutes at 30,000 X
gav' The pellet was resuspended in approximately half the volume of LIB
used for each resuspending step during lysis. Aggregates were removed
by centrifugation at 750 X gav for 10 minutes.
A Percoll (colloidal silica) gradient was obtained by mixing 22 mL
of the final supernatant with 15 mL of a mixture of 9 parts Percoll and
1 part 10-fold concentrated LIB, adjusted to pH 7.4 with HC1. The
mixture was centrifuged for 90 minutes at 40,000 X gav in a Sorvall SS-
82
Figure 2-3. Purification of Lysosomes on Percoll Gradients. All steps
were performed at 00-4oC. Cells were resuspended at 2 x 107 cells/rnL in
LIB. Lysosomes were purified according to Harms et al. (1981), with
modifications as indicated in the text.
Final 750 X 9 pellet
Cells in LIB Homogenize. then 750 X g. 15 min.;
Combined 750 X 9 supernatants I Filter, then 30.000 X g. 30 min .
if • 30,000 X 9 pellet 30,000 X 9 supernatant
t Resuspend in 1/2 volume LIB, then 750 X 9 centrifugation
t Pellet (discard)
I +
Supernatant
t Percoll suspension,
(15 mL 9:1 Percoll:10x LIB + 22 mL supernatant)
I 40.000 X g, 90 minutes
t • Lower 12 mL diluted Remainder (discard) 1:5 with LIB
t I 40.000 X g. 20 min . • Cloudy Layer - Supernatant
(discard) Continue 1:5 dilutions until material pellets
Pellet (discard)
t 40.000 X g. 20 min.
Final Pellet Resuspend in LIB;
480 X g. 10 min.
Supernatant (Purified Lysosomes)
.. 30.000 X g, 30 min.
Lysosomal Pellet
84
34 fixed-angle rotor and allowed to coast to a stop. The probe of a
Buchler Auto Densi-Flow was lowered to the bottom of the tube. The
lower 12 mL of the gradient was removed and diluted 1:5 with LIB.
After centrifugation at 40,000 X gav for 20 minutes, the clear
supernatant was removed by vacuum aspiration and discarded. The
remaining suspension was diluted with 5 volumes of LIB, followed by
centrifugation at 40,000 X gav for 20 minutes. This wash step was
repeated 4-5 times, until most of the organelles sedimented as a
pellet, indicating that most of the colloidal silica was removed. The
final pellet was resuspended in 2-4 mL of LIB and centrifuged at 480 X
gav for 10 minutes. The supernatant from this final centrifugation,
containing the purified lysosomes, was used for additional studies. If
lysosomes were needed in solid form, the sample was re-pelleted at
30,000 X gav for 30 minutes.
II.F. Lysosomal Membrane Protein and Phospholipid Analysis
Phospholipid content of lysosomal membranes was determined using
the method of Mrsny et al. (1986). Phosphate standards were prepared
using 0-200 nmol NaH2P04 ·H20. Cell samp"ies contained 5 x 106 cell
equivalents of purified lysosomes. Typically, 10-15 mg protein was
isolated from 109 cell-equivalents of lysosomal membranes. Samples and
standards were adjusted to 50)aL with deionized distilled water, and
centrifuged at highest speed in an Eppendorf microcentrifuge to
separate lysosomal membranes from intralysosomal fluid. After the
85
addition of 0.5 mL concentrated perchloric acid, each tube was
incubated for 1 hr. at 1300 C in a Temp-Blok module heater (Lab-Line
Instruments, Melrose Park, IL). Following phospholipid digestion, 3.0
mL water, 1.0 mL 2.5% (w/v) ammonium molybdate and 0.5 mL freshly
prepared 10% ascorbic acid were added. The phosphomolybdate color was
allowed to develop for 90 minutes in a 370 C water bath. The absorbance
of standards and samples was read at 820 rum against a water blank
carried through the entire procedure.
II.G. Lysosomal Transport Studies
II.G.1. Determination of Lysosomal Sialic Acid
Lysosomes were prepared, as shown in Figure 2-3. Lysosomal Sia
was determined, as shown in Figure 2-4. The 480 X gav supernatant,
containing the purified lysosomes, (Figure 2-3) was centrifuged at
30,000 X gav for 30 minutes to pellet the lysosomes and remove residual
LIB. Lysosomes were ruptured in 0.5 mL deionized distilled water.
Separation of lysosomal contents from the membranes was performed by
centrifugation at 15,000 X gav for 15 minutes, as this force will
pellet lysosomes in aqueous buffers (Miyagi and Tsuiki, 1984). The
supernatant, containing the intralysosomal fluid, was run over a Biogel
P-2 column (200-400 mesh, 1 cm x 28 cm, equilibrated in deionized
distilled water) to separate macromolecules from low molecular weight
molecules. Eighty fractions (one mL each) were collected. The protein
peak (void volume) was identified using the protein assay described
86
Figure 2-4. Purification of Lysosomal Sialic Acid. Lysosomes were
purified, according to Figure 2-3, and subjected to hypotonic
disruption (Miyagi and Tsuiki, 1984). Intralysosomal free Sia was
determined with the thiobarbituric acid assay (Warren, 1959).
Percoll-Purified Lysosomal Pellet
~ waler,lhen 15,000 X g, 15 m;n,
Pellet Supernatant (lysosomal membranes) (lysosomal contents)
+ Lyophilize
+ Gel Filtration Column
A Protein in Void Volume
60 mL beyond Void Vol.
+ Pool and Lyophilize
~ 3 Successive QAE+ Anion . Exchange Columns
Washed in 10 mM NaCI
+ Sialic Acid Elution
With 1.0 M NaCI
Pool and Lyophilize
1.0 M NaCI Fractions for
Usa in TeA Assay
88
above. All non-protein fractions (up to fraction 80) were pooled and
lyophilized.
The dried material was resuspended in deionized distilled water,
in preparation for ion exchange chromatography. Separation of charged
and neutral sugars was performed using quaternary aminoethyl (QAE+)
anion exchange resin (Bio-Rad, Richmond, CA). Columns were prepared in
10 mL syringes equipped with 18 gauge needles and 20 cm Tygon tubing
(inner diameter = 1/32 in.). 6-8 mL of packed resin, pre-equilibrated
in 10 roM NaCl, was used in each syringe. Lysosomal samples were run
over 3 successive 5 mL Cellex QAE+ columns equilibrated in 10 roM NaCl.
The Sia was eluted from the columns with 10 mL 1.0 M NaCl per column.
The eluates from all three columns were pooled, lyophilized, and
analyzed for Sia content using a modified thiobarbituric acid assay
(Warren, 1959), in which a-acetyl groups are hydrolyzed prior to
analysis. A known mixture of sucrose, Glc, and Sia yielded 96.4%
recovery of Sia in the pooled 1.0 M fractions and 97.1% recovery of Glc
in the pooled 10 roM fractions (Glc analyzed by the P~rk-Johnson
ferricyanide assay; Spiro, 1966).
II.G.2. Distribution of Radioactivity Following [6-3Hl-ManNAC Incubation
Cells were grown for two days in DMEM/HS/PS containing 5~Ci/mL
[6- 3Hl-ManNAc (specific activity 30 Ci/mmol). Cells were fractionated
according to Figure 2-3, and aliquots of each fraction were assayed for
~-galactosidase activity, as described above. Scintillation counting
was performed on each sample using a Beckman LS-7000 liquid
89
scintillation counter. The lysosomes were subjected to hypotonic
rupture in distilled water and centrifuged at 15,000 X gav for 15
minutes. This supernatant and the cytoplasmic fraction were brought to
70% saturation in ammonium sulfate at RT to precipitate
(glyco)proteins. After centrifugation at 30,000 X gav (30 min., RT),
the supernatants were separated into neutral and negatively charged
molecules on Cellex QAE+ columns, as described in "Determination of
Lysosomal Sialic Acid". The radioactivity in each sample was
determined by scintillation counting.
II.G.3. Lysosomal Stability as a Function of Time
Lysosomes were purified, according to Figure 2-3, except that the
480 X g av supernatant was divided into 5-6 aliquots prior to the last
30,000 X gav centrifugation. The LIB was adjusted to pH 7.4 at 37oC.
The final lysosomal pellet in each aliquot was gently resuspended in 1
mL LIB (37oC, pH 7.4). Each of the samples was incubated at 37°C for
the desired time interval, and then centrifuged at 30,000 X gav for 30
minutes at RT. The resulting pellet and supernatant were assayed for
~-galactosidase activity to determine the extent of lysosomal rupture.
II.G.4. Release of (3H)-Sialic Acid from Lysosomes
Cells were grown for two days in DMEM/HS/PS containing 5}lCi/mL
[6-3H]-ManNAc (specific activity 30 Ci/mmol). Cells were fractionated
according to Figure 2-3, and fractions were assayed for 3H content and
~-galactosidase activity, as described above. Incubation of the
90
purified lysosomes at 370 C was performed, as described above. Aliquots
of each pellet and supernatant were assayed for ~-galactosidase
activity to determine the extent of lysosomal rupture. Scintillation
counting was used to measure the radioactivity in each pellet and
supernatant sample.
II.G.s. Accumulation of Lysosomal Cystine
Cells were grown for 3 days, washed in RT-PBS, and resuspended at
1.0 X 107 cells/mL in RT-OMEM (no HS/PS) with 1.0 roM cystine dimethyl
ester (COME; Smith et al., 1987). After incubation at 370 C for 20
minutes, the cells were washed twice in RT-PBS. Lysosomes were
prepared as in Figure 2-3. Intralysosomal cystine was determined
according to Figure 2-4, except that the procedure was terminated after
lyophilization of the non-protein fractions from gel filtration.
Cystine analysis was performed using modifications of a
spectrophotometric assay. Cystine-containing samples or standards (0.5
mL each) were reduced to cysteine with 1 mL 2 roM dithiothreitol
(Clelland, 1964). The excess dithiothreitol was removed by chelation
to Na arsenite (Jocelyn, 1972). The cysteine was then assayed with
dithionitrobenzoic acid (Jocelyn, 1987).
91
Table 2-1. Reagents and Suppliers
Reagent
Acetic acid
Ammonium molybdate
Ammonium sulfate
Ascorbic acid
Bio-Gel P-2, 200-400 mesh
BSA
Calcium chloride
CDME
Cellex QAE+ resin
CUpric sulfate
DMEM
Dithionitrobenzoic acid
Dithiothreitol
EDTA
Folin-Ciocalteu Reagent
Glc
GlcNAc
Gly
HS
Hydrochloric acid
Magnesium chloride
ManNAc
Supplier
Fisher Scientific, Fair Lawn, NJ
Fisher
Mallinckrodt, st. Louis, MO
Sigma, st. Louis, MO
Bio-Rad, Richmond, CA
Sigma
Baker, Phillipsburg, NJ
Sigma
Bio-Rad
Mallinckrodt
Gibco, Grand Island, NY; Cat. # 430-1600
Sigma
Sigma
Mallinckrodt
Sigma
Baker
Sigma
Sigma
Gibco; Cat. # 230-6050
Fisher
Mallinckrodt, St. Louis, MO
Sigma
------~----~---- ~ ---------
Methanol
4-MU and 4-MU conjugates
5-NAcNeu
NP-40
PBS
Penicillin (in PS)
Perchloric acid
Percoll
Phosphoric acid
Potassium ferricyanide
Potassium ferrocyanide
Sodium acetate
Sodium arsenite
Sodium carbonate
Sodium chloride
Sodium citrate
Sodium hydroxide
Sodium meta periodate
Sodium phosphate (dibasic)
92
New England Nuclear, Wilmington, DE
Baker
Sigma
Sigma
Particle Data Laboratories, LTD, Elmhurst, IL
Gibco; Cat. # 450-1400
Eli Lilly, Indianapolis, IN; vial #526
Mallinckrodt
Sigma
Fisher
Fisher
Fisher
Fisher
Sigma
Fisher
Fisher
Baker
Fisher
Fisher
Mallinckrodt
Sodium phosphate (monobasic) Baker
Sodium potassium tartrate Mallinckrodt
Sodium sulfate Allied Chemicals, Morristown, NJ
Sodium taurocholate Sigma
streptomycin (in PS)
Sucrose
Sulfuric acid
Thiobarbituric acid
Triethanolamine
Tris (Trizma Base)
Triton X-100
Trypan blue
WGA
93
Eli Lilly, vial # 431
Sigma
Fisher
Aldrich, Milwaukee, WI
Baker
Sigma
Calbiochem, Los Angeles, CA
Eastman Kodak Co., Rochester, NY
Bethesda Research Laboratories, Bethesda, MD
-------~- ~---~~-~ ~---~
Chapter Three
Results
III.A. In Vitro Cell Growth
EL4 and WB6 cells had different patterns of cell density when
grown in tissue culture. The experiments described below were performed
either two or three days after dilution of cell suspensions with fresh
DMEM/HS/PS. Two days after dilution, EL4 cells were at (5 ~ 2) x 105
cells/mL. One day later. the cells were at (8 ! 2) x 105 cells/mL. WB6
cells were at (4 + 1) x 105 cells/mL two days after dilution, and (1.1
~ 0.2) x 106 cells/mL after three days. EL4 cells were at 63% of their
maximum density two days after dilution, while WB6 cells were at only
36% of their maximum density at that time. In addition, the density of
EL4 cells was slightly greater than that of WB6 cells two days after
dilution. However, the density of WB6 cells was greater than that of
EL4 cells three days after dilution. The terms "day 2" and "day 3"
will refer to cells used either two or three days, respectively,
following dilution with fresh DMEM/HS/PS.
94
95
III.B. Preliminary 4-MU Studies
III.B.1. 4-MU Fluorescence as a Function of pH and Temperature
4-methylumbelliferyl conjugates were used to assay a variety of
lysosomal hydrolases. The enzyme reaction product 4-MU has an ionizable
phenolic hydroxyl group (Figure 3-1). The conjugate base has a much
higher fluorescence intensity than the unionized form, due to the
participation of the unpaired electrons on the phenolic oxygen atom in
the ring resonance. Fluorescence is therefore dependent on pH (Table
3-1). A plot of fluorescence as a function of pH is seen in Figure 3-2.
The data in Table 3-1 were used to calculate the pKa of the
phenolic proton. solution of the Henderson-Hasselbach equation gives a
pKa value of 8.8. The gly-carb buffer used to stop the enzyme reaction
should therefore be at pH 9.8 or higher to ensure that ~ 90% of the
molecules are ionized and fluorescence is at its maximum. However,
high pH tends to increase base-catalyzed substrate hydrolysis, so the
gly-carb was prepared to have a pH of 9.4 at OOC. Since the pH of
glycine buffers is highly temperature-sensitive, 4-MU fluorescence is
indirectly affected by temperature. Whenever possible, the same bottle
of gly-carb was used for all samples and standards in a given
experiment to avoid any pH variations between bottles. In addition,
all 4-MU standards were prepared under the same conditions of pH and
ionic strength as the samples.
96
Figure 3-1. Ionization of 4-MU. The phenolic proton has a pKa of 8.8,
as calculated using the Henderson-Hasselbach equation.
r0 I U
r0 I U
I.(')
en I
lQ CO I a.
o
o I
o I
98
Table 3-1. Dependence of 4-MU Fluorescence ~ Eli
pH Rel. % Fluor
6.0 1.2
7.0 5.8
8.0 8.1
8.5 12.8
9.0 69.8
9.3 81.4
9.6 90.7
9.9 93.0
~0.2 100.0
Samples containing 6 nrnoles 4-MU in 3.18 mL of 85 roM glycine were titrated to the indicated pH using 1.0 M Na carbonate. Fluorescence was corrected for sample dilution. The pH 10.2 sample was assigned a relative percent fluorescence of 100%, as described in Materials and Methods. The fluorescence of the remaining samples was determined relative to the pH 10.2 sample.
99
Figure 3-2. 4-MU fluorescence versus pH. The data in Table 3-1 were
fitted to the Henderson-Hasselbach equation. The circles are the
actual data points and the curved line represents the theoretical
curve, with pKa = 8.8.
=
0 Q
=> ::2E cn:I: V c.. IJ..W oU 0
Z WW UU coZ zCJ) 0 IJJIJJ 00:: ~ ZO => IJJ=> -' 0.-' ~g IJJIJ.. 0
:I: Co
o
3:)N3::>S3C10nl.:l lN3::>Cl3d 31\11V'l3C1
101
III.B.2. Design of Hydrolase Activity Experiments
Neuraminidase, j3-galactosidase, ~-mannosidase, acid phosphatase,
and acetylesterase activities were studied in the linear region of a
plot of nmoles 4-MU released versus time. Neuraminidase and acid
phosphatase activities were corrected for spontaneous substrate
hydrolysis using heat-inactivated controls. An example of this is seen
in Figure 3-3. The slope of this line is nmoles 4-MU product/hour.
Total activity for the sample was calculated from the slope, as
follows:
Total activity = 109 cells
nmoles/hour x total sample volume aliquot volume
total # cells in sample
Most experiments were performed with detergent-solubilized samples,
because the majority of the lysosomal enzymes studied in this work are
membrane-associated (Holtzman, 1989). No additional enzyme activity was
released with detergent concentrations higher than 0.5%, and there was
no measurable enzyme activity in the insoluble membrane pellet
following detergent solubilization and centrifugation (data not shown).
IILC. Studies on RSB-Prepared "Lysosomes"
III.C.1. Distribution of Lysosomal Activity
Preliminary results using a number of lysosomal marker enzymes
indicated a high degree of lysosomal rupture in the hypotonic buffer
used for N2 cavitation. (T. Keller, personal communication, 1988).
102
Figure 3-3. Typical neuraminidase assay results. Neuraminidase assays
were performed using normal and heat-inactivated samples, as the
substrate 4-MU-Sia undergoes significant spontaneous hydrolysis.
Samples were prepared, as described in the "Neuraminidase Activity"
section in Materials and Methods.
The average values of experimental (~) and heat-inactivated
control (GJ) samples are shown for three time points. The lower line
(.) is the difference between the experimental and control values,
representing substrate hydrolysis due to the enzyme. Statistical
analysis of these results can be seen in Table 3-4.
------_. __ ._ .. _-_.- _ .. _ .. _--_ ....
EL4 Neuraminidase Activity
2~--------------------------------~
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Time, Hours
----- ------.. -----.- - - _ .. _ ... _---
104
~-galactosidase was used to follow lysosomal activity in the
purification scheme in Figure 2-1. Enzyme assays were performed as
described in Materials and Methods, using 4-MU-Gal as the substrate.
Typical activity distributions for EL4 and WB6 are shown in Table 3-2.
The large percentage of "lysosomal" activity in both the cytoplasmic
fraction and the 11,000 X gav supernatant (see Figure 2-1) was
consistent with lysosomal rupture. This must be kept in mind when
examining data on RSB-prepared cell lysates. In this experiment, the
total activity in the initial EL4 cell lysate was 2.4 times higher than
it was for WB6.
III.C.2. Lysosomal NaCl Sensitivity
The data in Table 3-2 suggested that lysosomal rupture had
occurred during N2 cavitation. The RSB used to swell the cells
contained 10 roM NaCl, and we reasoned that this might be the problem.
The experiments shown in Table 3-3 were undertaken to see if modified
RSB, containing a higher NaCl concentration, might sufficiently swell
the cells without rupturing the lysosomes. Cells were swollen in RSB,
as described in Materials and Methods, except that the NaCl
concentration was varied from 10 roM to 100 roM, with other constituents
held constant.
Following N2 cavitation, the cell lysate was centrifuged at 300 X
gav' and the supernatant was centrifuged at 4000 X gav' This
centrifugal force was selected because it was the first point of
separation of lysosomes and cytoplasm in Figure 2-1. The resulting
lOS
Table 3-2. Typical Distribution of ~-Galactosidase Activity in Cells Fractionated in RSB.
EL4 WB6 Total Total
Activity Percent Activity Percent
Nuclei and un- 381 5% 189 6% broken cells
11,000 x 9 pellet 913 12% 684 22% (ltlysosomes ll
)
11,000 x 9 sup. 1370 18% 878 28%
20,000 x 9 pellet 304 4% 95 3% (PM, ER, Golgi)
20,000 x 9 sup. 4410 58% 1520 48% (cytoplasm)
Cell Lysate 7610 100% 3170 100%
Cells were fractionated, as described in Figure 2-1. Samples were assayed for ~-galactosidase activity using 0.5 roM 4-MU-Gal, as described in Materials and Methods. The numbers represent total nmoles 4-MU released per hour per 109 cell-equivalents. These values are expressed as a percent of the total activity measured in the initial cell lysate, prior to any centrifugation.
106
pellet and supernatant were assayed for ~-galactosidase activity, as
described in Materials and Methods. Percent intact lysosomes is
defined as:
activity in 4000 X gav pellet __ ~~ __ ~ ____________ ~_________________________ X 100
activity in 4000 X gav pellet + 4000 X gav supernatant
The results of these experiments are seen in Table 3-3 and Figure
3-4. The figure shows that the percent rupture of EL4 was relatively
independent of NaCl concentration, while WB6 lysosomal stability
increased with increasing NaCl concentration. The data for RSB with
100 roM NaCl were not shown, because fewer than 10% of the cells
ruptured following release from the N2 bomb. The vertical bars
represent the standard deviation of two experiments. No error bars are
shown when single experiments were done.
The ratio of f'-galactosidase activities in the (4000 X gav pellet
+ 4000 X gav supernatant) in EL4 to the same quantity in WBG was 0.85 +
0.17 in four separate experiments (Table 3-3). This ratio is lower
than the ratio of total activities in the cell homogenates (Table 3-2
and Table 3-5) because fewer EL4 cells ruptured following release from
the N2 bomb, resulting in a larger fraction of the total activity in
the 300 X 9 pellet (Figure 2-1).
These results appeared at first to contradict the results of Table
3-2. The 11,000 X gav pellet in EL4 contained a lower percentage of the
total ~-galactosidase activity than did WBG (Table 3-2). However,
these samples were obtained after the 4000 X gav centrifugation (Figure
107
Table 3-3. Sensitivity of 4000 x gav "Lysosomes" to [NaCI).
EL4
[NaC1) Pellet sup.
10 roM 2.60 5.56
2.20 5.41
25 roM 8.37 21.0
50 roM 2.84 8.32
1.57 5.37
% in Total Pellet
8.16 32%
7.61 29%
29.4 28%
11.2 25%
6.94 23%
Pellet
2.25
6.91
7.13
2.94
WB6 % in
Sup. Total Pellet
10.5 12.8 18%
23.5 30.4 23%
7.85 15.0 48%
3.52 6.46 45%
Cells were grown three days in vitro, then disrupted with N2 cavitation. The NaCI concentration in RSB was varied from 10 roM to 50 roM. The 4000 X gav pellet and 4000 X gav supernatant were assayed for # -galactosidase activity to determine the percent intact lysosomes. Values in columns 2, 3, 5, and 6 are total nmoles 4-MU released per hour per 106 cell-equivalents.
108
Figure 3-4. Sensitivity of 4000 X gav lysosomal samples to NaCl
concentration. Cells were grown in vitro for three days, then
disrupted with N2 cavitation. The NaCl concentration in RSB was
varied from 10 roM to SO roM. The 4000 X gav pellet and 4000 X gav
supernatant, as shown in Figure 2-1, were assayed for p-galactosidase
activity to determine the percent intact lysosomes (Table 3-3).
--------------- -------
en W :E 0 en 0 en ~ I-(.)
~ Z -~
SENSITIVITY OF LYSOSOMES
TO [Noel]
50 .-EL4 o-WB6 9""
40
30 -+--+-
20
10
0 10 20 30 40
[Noel] , mM
----- -------------- -------- - -
110
2-1). The data in Table 3-3 suggested that, of the material which
pelleted at 4000 X gav' a greater percentage of it remained intact in
WB6 during the 11,000 X gav centrifugation. The studies shown in Table
3-3 were done to backtrack to the first point of separation of
"1ysosomes" and cytoplasm in Figure 2-1. 4000 X gav data were not
collected in the experiments shown in Table 3-2, as the problem of
lysosomal rupture was not discovered until after these experiments were
performed.
III.C.3. Neuraminidase Activity
III.C.3.a. Results
Cells were disrupted using N2 cavitation in RSB. Lysosome
enriched fractions were prepared, as described in Materials and
Methods. Total activity is defined as nmol 4-MU released from 4-MU-Sia
in one hour by 109 cells. A variety of detergents were used at 0.5%
(w/v) final concentration. These results are shown in Table 3-4. The
total activity appeared to be a function of the detergent used
(Alhadeff and Wolfe, 1981), but may be partly due to pH effects of the
detergent that were not completely accounted for in the 4-MU standards
(see Figure 3-2). Neuraminidase is a particularly unstable enzyme whose
activity decreases rapidly over time (Spaltro and Alhadeff, 1984). The
interval between enzyme preparation and enzyme assay was not recorded,
and may very likely have contributed to the observed differences in
total activity. Despite these difficulties, the ratio of EL4
neuraminidase activity to that in WB6 was consistent from experiment to
------------------------
111
Table 3-4. Neuraminidase Activity in RSB-Prepared "Lysosomes"
Detergent Expt. # Total Activity/109 cell-equiv.
EL4 WB6 EL4/WB6
Triton X-100 1 24 + 78 9 + 13 2.7 - -Nonidet P-40 2 57 + 9 19 + 4 3.0 -Na Taurocholate 3 13 + 1 5 + 1 2.6 -Na Taurocholate 4 18 + 1 6 + 3 3.0 - -
Average Ratio 2.8 + 0.2 -
Neuraminidase activity was measured in 11,000 X gav pellets, as shown in Figure 2-1. Pellets were solubilized in 0.5% (w/v) detergent, and centrifuged to remove membrane-insoluble material'. The supernatants were used as the enzyme source in neuraminidase assays, as described in Materials and Methods.
The numbers in the first two activity columns represent the total nmoles 4-MU released from 4-MU-Sia in 1 hour by 109 cell-equivalents. The last column is the ratio of total activity in EL4 "lysosomes" to the total activity in WB6 "lysosomes", in a given experiment. The errors indicated were obtained, as described in the text. The number of degrees of freedom varied from 4 to 14, based on the F test, and the students' t value was selected for the 95% confidence interval (0.975 for a two-tailed test).
------ ._---_ .. _--- -
112
experiment, with an average value of 2.8 + 0.2.
III.C.3.b. Statistical Analysis of Neuraminidase Data
The high error in the neuraminidase data necessitated statistical
analysis to determine whether or not the calculated ratio of EL4
activity to that in WBG was significant. Three methods of statistical
analysis were used. Method one (equation 1, below) used the corrected
value at each time point to compute the slope in a graph of nmoles 4-
MU/mg protein versus assay time (in hours). Method two computed the
slope and its variability (equation 2) for the experimental points and
for the control points. The corrected slope was then calculated,
according to equation 3. Method three disregarded the individual data
points and slopes. Instead, this method focused on the calculation of
the ratio of total neuraminidase activity in EL4 to that in WBG. The
reliability of this ratio was calculated using equation 4. The total
activities in Table 3-4 were calculated using method two, and the
variability in the EL4/WBG ratio was calculated using method three.
Equation 1
Corrected nmoles/mg
= Expt'l Control nmol/mg nmol/mg
standard deviation standard deviation
Equation £
Variability = (SD of slope)2 = in slope
-----------------_ .. _-_ .. ------
of of
+ [( SDe)2 + ( SDc)2]1/2 -
the experimental points the control points.
(~i - Yi)2/n-2
(xi -x)2
113
where xi and Yi = individual x and y values, respectively x = average x value Yi = predicted y-value for a given x-value n = # items
Equation ~
Slope = Expt'l - Control + t(Var. e + var. c )1/2, where Slope Slope
Var. e = experimental variability (Eq. 2) Var. c = control variability
t = student's t value for a 95% confidence interval (CI) in a population with n degrees of freedom.
Equation 4
95% CI = Mean ~ t·SD/N1/ 2
where t = student's t value for n-l degrees of freedom
N # items in series.
III.C.4. f3-Galactosidase Activity
Activity was measured as for neuraminidase, except that the whole
cell lysate was used as the enzyme source. The substrate was 4-MU-Gal.
The results from five separate experiments can be seen in Table 3-5.
The total activities varied from one experiment to the next, and in
experiments using different detergents. However, the ratio of total
activities in the two cell lines was consistent. The average ratio of
total activity in EL4 to that in WB6 was 2.3 + 0.6.
114
Table 3-5. ~-Galactosidase Activity in RSB-Prepared Cell Lysates.
Total Activity/l06 cell-equiv.
Detergent ~! EL4 WB6 EL4/WB6
none 1 11.5 5.87 2.0
2 11.9 3.43 3.5
Na Taurocholate 3 3.72 1. 76 2.1
4 14.1 7.23 2.0
5 9.53 5.82 1.8
Average Ratio 2.3 + 0.6 -
~-Galactosidase activity was measured in cell lysates from N2 cavitation. Samples were untreated, or were solubilized in 0.5% (w/v) Na taurocholate, and centrifuged to remove membrane-insoluble material. The supernatants were used as the enzyme source in fjgalactosidase assays, as described in Materials and Methods. The numbers in the first two activity columns represent the total nmoles 4-MU released from 4-MU-Gal in 1 hour by 106 cell-equivalents. The last column is the ratio of total activity in EL4 to the total activity in WB6, in a given experiment.
115
III.C.S. oZ-Mannosidase Activity
Activity was measured like ~ -galactosidase, except that the
substrate was 4-MU-Man. Two separate experiments are shown in Table 3-
6. The ratio of O<-mannosidase activity in EL4 relative to that in WB6
was 3.1 + 0.3
III.D. Studies on Percoll-Prepared Lysosomes
III.D.1. Purification of Lysosomes on Percoll Gradients
The data in Tables 3-2 and 3-3 pointed to the need for an
alternative method of lysosome purification if the study of
neuraminidase was to continue. Many of the studies of lysosomal enzymes
published since the mid-1980's use Percoll density gradient
centrifugation to purify lysosomes. Percoll is a density gradient
medium designed to maintain physiological conditions throughout
centrifugation experiments. It is a sterile, colloidal suspension of
silica particles, 15-30 nm in diameter, coated with polyvinyl
pyrollidone. Percoll does not penetrate membranes, and so allows cells
and organelles to band isopycnically at their true buoyant densities.
The Percoll purification scheme used by Harms et al. (1981) was
modified for use with EL4 and WB6 lysosomes.
Lysosomes were purified, as in Figure 2-3. Fractions were
analyzed for ~-galactosidase activity using 4-MU-Gal. The
distribution of total ~-galactosidase activity for a typical
experiment is shown in Figure 3-5. The overall recovery of activity
116
Table 3-6. o(-Mannosidase Activity in RSB-Prepared Cell Lysates.
Total Activity/lOG cell-equiv.
Detergent EL4 WBG EL4(WBG
Na Taurocholate 1 0.799 0.237 3.4
2 1.98 0.700 2.8
Av. = 3.1 + 0.3
o\-Mannosidase activity was measured in cell lysates from N2 cavitation. Samples were solubilized in 0.5% (w/v) Na taurocholate and centrifuged to remove membrane-insoluble material. The supernatants were used as the enzyme source in o(-mannosidase assays, as described in Materials and Methods. The numbers in the first two activity columns reEresent the total nmoles 4-MU released from 4-MUMan in 1 hour by 10 cell-equivalents. The last column is the ratio of total activity in EL4 to the total activity in WBG, in a given experiment.
117
Figure 3-5. Distribution of EL4 f5-Galactosidase activity in LIB
Prepared Cells. Cells were fractionated, as shown in Figure 2-3, and
assayed for ~ -galactosidase activity, as described in Materials and
Methods. The values shown are the percent of total activity in a given
sample, relative to the initial cell lysate.
Distribution of EL4 ~ -Galactosidase Activity in LIB-Prepared Cells
+ Cell Lysate (100%)
I 750 X g, 4 times
final 750 X g pellet (11.5%)
+ combined 750 X g
supernatants I 30,000 X 9
+ Pellet (67.9%) Supernatant (9.5%)
Resuspend, then 750 X 9
Pellet (aggregates) (4.9%)
Supernatant (34.7%)
I Percoll Gradient
Lower 12 mL (20.7%) 40,000 X 9 washes
Protein Fraction (5:9%)
Purified Lysosomes
(13.4%) Wash Solutions
(4.0%)
119
was 49.2%, but this value excluded unassayed portions of the Percoll
gradient. The percent activity in the 750 X gav pellet (unbroken cells
and nuclei) was the most variable, and may have been affected by the
age and/or health of the cells, as well as variations in the force with
which the cells were homogenized.
The degree of purification, as measured by the increase in
specific activity during the purification scheme, is indicated in Table
3-7. The 750 X gav supernatant had a specific activity 2.61 times
higher than the cell homogenate, due to removal of nuclei and nuclear
proteins. Removal of the cytoplasmic proteins in the 30,000 X gav
centrifugation resulted in a pellet with a specific activity 4.87 times
higher than the cell homogenate. The purified lysosomes, separated from
other organelles on the Percoll gradient, had a specifc activity 10.7
times higher than the cell homogenate.
III.D.2. Neuraminidase Activity
Lysosomes were prepared, as described in the Materials and Methods
section, "Purification of Lysosomes in Isotonic Sucrose," (outlined in
Figure 2-3). Normal and heat-inactivated lysosomal samples were
solubilized in 0.5% Na taurocholate and assayed for neuraminidase
activity as in Figure 3-3, using 0.5 mM 4-MU-Sia. The results shown in
Table 3-8 were corrected for lysosomal rupture, as measured by the
distribution of acid P-ase activity in the 30,000 X gav samples:
Corrected Neuraminidase = ~ ______ ~M~e~a~s~u~r~e~d~A~c~t~i~v~i~t~y _________ __ Activity Fraction of Intact Lysosomes
120
Table 3-7. Enhancement of ~-Galactosidase Specific Activity During EL4 Lysosome Purification
Specific Activity, Relative Sample nrnole/mg-hour Specific Act.
Cell Lysate 64.1 1.00
750 X gav 167 2.61 Supernatant
30,000 X gav 312 4.87 Pellet
Percoll-Purified 697 10.9 Lysosomes
Cells were fractionated, as shown in Figure 2-3, and assayed for protein and ~-galactosidase activity, as described in Materials and Methods. Specific activity values are nrnoles 4-MU released per mg protein per hour. The relative specific activity was calculated by dividing a given specific activity by the specific activity of the cell lysate.
121
Table 3-8. Neuraminidase Activity in LIB-Prepared Lysosomes from EL4 and WB6.
% Intact Corrected Total Neur. Activit:£ L:£sosomes Neur. Activ.
Days After Dilution EL4 WB6 EL4 WB6 EL4 WB6
2 20.4 21.1 10.4 20.9 196 111
3 15.8 12.6 74.8 48.3 21.1 26.1
Neuraminidase activity was measured in purified lysosomes, as shown in Figure 2-3. Samples were solubilized in 0.5% (w/v) Na taurocholate, and centrifuged to remove membrane-insoluble material. The supernatants were used as the enzyme source in neuraminidase assays, as described in Materials and Methods. The numbers in the "Total Neuraminidase Activity" columns represent the total nmoles 4-MU released from 4-MU-Sia in 1 hour by 109 cell-equivalents. Columns 4 and 5 are the percent intact lysosomes, as measured by ~-ga1actosidase activity. Correction for lysosomal rupture, as described in text, is shown in columns 6 and 7.
-----------------------------
122
The differences in the total activity in the two experiments could be
due to factors discussed previously for RSB-prepared "lysosomes", or
there may be real differences in the activity of this enzyme two days
after dilution, relative to the activity after three days. The total
activity in EL4 also seems to depend on the density of the cells.
Two days after dilution, the ratio of EL4 lysosomal neuraminidase to
that of WB6 was 1.76. After three days, both activities decreased, but
EL4 decreased more, resulting in WB6 having more neuraminidase activity
than EL4. The ratio of EL4 activity to WB6 activity was 0.808.
In addition, the percent intact lysosomes also varied between cell
lines on day 2 and day 3. Two days after dilution, the ratio of percent
intact lysosomes in EL4 to that of WB6 was 0.50. After three days, the
same ratio was 1.5. EL4 lysosomes were 7.2-fold more stable on day
3 than on day 2. For WB6 cells, this quantity was 2.31. Several
possible interpretations of this data will be examined in Chapter four.
IILD.3. ~ -Galactosidase Activity
Whole cell lysates were solubilized using 0.5% Na taurocholate and
assayed for ~ -galactosidase activity using 0.5 mM 4-MU-Gal. The
results of three such experiments are shown in Table 3-9. The total
activities varied as they had in previous experiments. The ratio of
total ~-galactosidase activity in the two cell lines was dependent on
the number of days after dilution with fresh medium. EL4 had 1.37 times
more activity than WB6 on day two, and on day three the ratio of
EL4av/WB6av was 0.9 ~ 0.1.
123
Table 3-9. Total~-Galactosidase Activity in LIB-Prepared Lysates of EL4 and WB6.
Total Activity/106 cell-equiv.
Days After ~! Dilution EL4 WB6 EL4!WB6
1 2 14.9 10.9 1.37
2 3 8.15 9.90 0.823
3 3 14.0 13.5 1.04
~-Galactosidase activity was measured in LIB-prepared cell lysates, as described in Materials and Methods. Samples were solubilized in 0.5% (w/v) Na taurocholate, and centrifuged to remove mernbraneinsoluble material. The supernatants were used as the enzyme source in ~-galactosidase assays, as described in Materials and Methods. The
numbers in the first two activity columns represent the total nrnoles 4-MU released from 4-MU-Gal in 1 hour by 106 cell-equivalents. The last column is the ratio of activity of EL4 to that of WB6, for each experiment.
-------------------- -
124
III.D.4. Acetylesterase Activity
Samples were prepared in LIB, as described in Materials Methods,
and assayed for acetylesterase activity using 1.0 roM 4-MU-Ac. The
results of these experiments are shown in Table 3-10. Each of these
experiments was done on cells three days after dilution. Unlike other
acid hydrolases, these results were consistent from one experiment to
the next. In addition, the total acetylesterase activity in EL4 was
greater than that in WBG (average ratio = 1.8 ~ 0.2), whereas in the
other acid hydrolases investigated, the total activity in WBG was
higher than EL4 on day 3. For example, the ratio of EL4 neuraminidase
activity to WBG neuraminidase activity on day 3 was 0.808 (Table 3-8).
Similarly, the average ratio of ~-galactosidase activity in the two
cell lines was 0.9 (Table 3-9).
III.D.S. Acid Phosphatase Activity
Whole cell lysates were assayed for acid P-ase activity using 1 roM
4-MU-P, as described the "Acid Phosphatase Assay" section in Materials
and Methods. A number of experiments are shown in Table 3-11, grouped
by number of days after dilution with fresh medium. The cells had
somewhat more acid phosphatase activity than they did of the other acid
hydrolases studied. The ratio of acid P-ase activities between the two
cell lines was dependent on the number of days after dilution,
regardless of the actual activity in each cell line. On day 2, EL4 had
more acid P-ase activity than WBG (ratio = 1.4 ~ 0.3). On day 3, the
ratio was 0.79 + 0.09.
125
Table 3-10. Total Acetylesterase Activity in LIB-Prepared Cell Lysates of EL4 and WB6.
Total Activity/106 Cell-equiv.
Days After ~! Dilution EL4 WB6 EL4/WB6
1 3 30.5 15.1 2.02
2 3 26.9 17.0 1.58
3 3 27.2 15.6 1. 74
Average Ratio 1.8 + 0.2 -
Acetylesterase activity was measured in LIB-prepared cell lysates, as described in Materials and Methods. Samples were solubilized in 0.5% (w/v) Na taurocholate, and centrifuged to remove membrane-insoluble material. The supernatants were used as the enzyme source in acetyl esterase assays, as described in Materials and Methods. The numbers in the first two activity columns represent the total nmoles 4-MU released from 4-MU-Ac in 1 hour by 106 cell-equivalents. The last column shows the ratio of activity of EL4 to that of WB6, for each experiment.
126
Table 3-11. Total Acid Phosphatase Activity in LIB-Prepared Lysates of EL4 and WB6.
Total Activity/106 cell-equiv.
Days After Expt. # Dilution EL4 WB6 EL4/WB6
1 2 29.7 23.8 1.25
2 2 52.3 41.4 1.26
3 2 30.8 30.5 1.01
4 2 41.1 25.7 1.60
5 2 44.0 22.6 1.95
6 2 39.0 25.4 1.54
Average 2 40 + 8 28 + 6 1.4+ 0.3
*******************************************************************
7 3 25.1 37.4 0.671
8 3 22.7 25.1 0.904
9 3 29.1 38.1 0.764
10 3 27.1 32.7 0.829
Average 3 26 + 2 33 + 5 0.79 + 0.09
Acid phosphatase activity was measured in LIB-prepared cell lysates, as described in Materials and Methods. Samples were solubilized in 0.5% (w/v) Na taurocholate, and centrifuged to remove membraneinsoluble material. The supernatants were used as the enzyme source in acid phosphatase assays, as described in Materials and Methods. The numbers in the first two activity columns represent the total nmoles 4-MU released from 4-Mli-P in 1 hour by 106 cell-equivalents. The third activity column indicates the ratio of activities of EL4 and WB6, for each experiment. The last column shows the average ratio, plus or minus its standard deviation, for the indicated number of days after dilution with fresh medium.
127
III.D.G. Analysis of Phospholipid and Protein in Lysosomal Membranes
The variation in the ratios of acid P-ase activity in EL4 and WBG
may result from different numbers and/or sizes of lysosomes in the two
cell lines at various points in their growth cycle (T. Keller, personal
communication, 1988). This question was addressed by determining the
amount of lysosomal membrane phospholipid and of lysosomal membrane
protein using lysosomes prepared in LIB, as described in Materials and
Methods. Separate experiments were performed two days and three days
after dilution of cells, since lysosomal acid P-ase activity was
dependent on the state of growth of the cells (see Table 3-11).
The results of lysosomal phospholipid and lysosomal protein
analysis are shown in Table 3-12. The individual values of phospholipid
and protein are not shown, since the data was not corrected for
lysosomal rupture. However, a comparison can be made of the ratio of
these two quantities in EL4 and WBS. The average ratio of lysosomal
membrane phospholipid to lysosomal membrane protein was 2.G3 ! 0.03 two
days after dilution, regardless of cell line. In contrast, the ~esults
on day 3 varied between the cell lines. In EL4, the ratio umol
phospholipid/mg protein was 2.59 ! 0.01, while in WBG this ratio was
2.99 + 0.03.
128
Table 3-12. Analysis of Phospholipid and Protein in Lysosomal Membranes.
~ole EhosEholiEid mg Erotein
Days After Dilution Expt. # EL4 WB6 EL4/WB6
2 1 2.60 2.70 0.963
2 2.70 2.50 1.08
Ave. 2.65 2.60 1.02 + 0.06 -
3 1 2.60 2.96 0.878
2 2.58 3.02 0.854
Ave. 2.59 2.99 0.866 + 0.01 -
LIB-prepared lysosomes were purified, according to Figure 2-3. Lysosomes were ruptured in deionized water and centrifuged at 15,000 X gav to pellet membranes. Membrane phospholipid and protein were analyzed spectrophotometrically, as described in Materials and Methods. The first two data columns represent two separate experiments for EL4 and WB6, respectively. The last column is the value for EL4 divided by that of WB6.
--------------.--- --------.-.-
129
III.E. Lysosomal Transport Studies
III.E.1. Lysosomal Accumulation of Sialic Acid
To determine whether or not Sia transport was defective in W86,
cells were cultured with 50 roM ManNAc in DMEM/HS/PS. ManNAc is the
metabolic precursor of Sia (Figure 1-10), and virtually all the
radioactivity in cells cultured in the presence of [6-3H]-ManNAc is
recovered as Sia (Diaz and Varki, 1985).
EL4 and W86 were grown in the presence of 50 roM ManNAc for either
two or three days after dilution with fresh DMEM/HS/PS. This comparison
was necessary, as earlier studies (Tables 3-8, 3-9, and 3-11) indicated
that the total lysosomal activity, and therefore possibly the total
number of lysosomes, was dependent on the state of growth of the cells.
Cells were assayed for lysosomal acid P-ase activity and for
lysosomal free Sia, as described in the Materials and Methods. After
correcting for lysosomal rupture and aliquot removal, the nmoles of Sia
per 106 cells was divided by the total units of acid P-ase activity per
106 cells to get a measure of free Sia per lysosomal equivalent. The
results of two separate experiments, each done for EL4 and W86 with and
without ManNAc, are shown in Table 3-13. The average value of nmol
Sia/unit acid P-ase for each cell line is examined further in Table 3-
14. The result for cells incubated with ManNAc is divided by the result
for the same cells without ManNAc, to determine the relative
accumulation of Sia in cells incubated with ManNAc.
The data in Table 3-13a indicate that on day 2, W86 had 1.S-fold
130
Table 3-13. Lysosomal Accumulation of Sia in EL4 and WBG.
lSia analysis and acid phosphatase assays were performed, as described in Materials and Methods.
2EL4-M = EL4 + 50 roM ManNAc in growth medium; WBG-M = WBG + 50 roM ManNAc in growth medium.
3Corrected for aliquot removal and lysosomal rupture, as measured by acid phosphatase activity distribution.
4nmol 4-MU released from 1 roM 4-MU-phosphate per hour per lOG cells.
131
Table 3-13. Lysosomal Accumulation of Sia in EL4 and WB6. 1
a. Lysosomes Purified Two Days After Cell Dilution.
Expt. EL4 EL4-M2 WB6 WB6-M2
Corr. nmol 1 3.63 3.25 3.43 10.5 Sia/106 cells3 2 2.87 2.90 3.21 10.1
Total Acid 1 39.0 36.4 25.4 25.7 P-ase4 2 44.0 42.0 22.6 20.8
**********************************************************************
nmol Sia 1 .0825 .0774 .152 .505 Unit Acid P-ase 2 .0737 .0796 .126 .488
Ave. .0781 .0785 .139 .497
b. Lysosomes Purified Three Days After Cell Dilution.
Expt. EL4 EL4-M2 WB6 WB6-M2
Corr. nmol 1 2.77 3.12 3.38 7.91 Sia/106 cells3 2 2.80 2.75 3.25 9.04
Total Acid 1 27.1 23.0 32.7 31.9 P-ase4 2 25.1 22.7 37.4 33.4
**********************************************************************
nmol Sia Unit Acid P-ase
1 2
Ave.
.102
.112
.107
.136
.121
.129
.103 .0869
.0950
.248
.271
.260
132
Table 3-14. Relative Accumulation of Sialic Acid.
nmol Sia/Unit Acid P-ase Activity
Day Two
Day Three
~1 EL4
1.01
1.20
WB6-M2 WB6
3.58
2.74
These data were calculated from Table 3-13. The average nmoles Sia/unit acid P-ase for the cells grown in 50 mM ManNAc (Table 3-13) was divided by the same value for the untreated cells. The data in column two are for EL4 cells grown either two days or three days following dilution with fresh DMEM/HS/PS. The data in column three are for WB6 cells grown either two days or three days in vitro.
1EL4 + 50 mM ManNAc in growth medium.
2WB6 + 50 mM ManNAc in growth medium.
133
more endogenous Sia per unit of lysosomal acid P-ase activity than did
EL4. This was more pronounced (6.3-fold more in WB6) in cells treated
with 50 roM ManNAc. On the average at day 3, EL4 had slightly more
endogenous Sia per unit of lysosomal acid P-ase activity than did WB6
(ratio = 1.1; Table 3-13b), but this value decreased when the cells
were cultured in SO roM ManNAc (EL4-M / WB6-M = 0.5; Table 3-13b). The
acid P-ase results are consistent with those of several other
experiments, and are included in Table 3-11 as well as as Table 3-13.
Table 3-14 is a comparison of untreated cells to the same cells
cultured in the presence of 50 roM ManNAc. On day 2, nmol Sia/unit acid
P-ase was the same in EL4 regardless of growth conditions. In contrast,
WB6 showed a 3.s8-fold increase in nmol Sia/unit acid P-ase when grown ,
in the presence of ManNAc. The results were slightly different three
days after dilution. The ratio EL4-M/EL4 increased from 1.01 (day 2) to
1.20 (day 3), while the ratio WB6-M/WB6 decreased from 3.78 to 2.74.
III.E.2. WB6 Lysosomal Sialic Acid Accumulation in Varied Concentrations of ManNAc
To determine the effect of ManNAc concentration on WB6 lysosomal
Sia accumUlation, cells were incubated for three days in the presence
of 0 roM, 10 roM, 25 roM, or SO roM ManNAc. Acid P-ase and Sia
determinations were as described in Table 3-13. The results of these
experiments are shown in Table 3-15 and Figure 3-6. The ratio of nmol
Siajunit acid P-ase was consistent between the two experiments. Table
3-15 shows that WB6 cells cultured in SO roM ManNAc had an average of
2.3 times more lysosomal Sia per unit acid P-ase than did cells
134
Table 3-15. WB6 Lysosomal Sialic Acid Accumulation as a Function of ManNAc Concentration. 1
ManNAc Concentration in Growth Medium
Exet. OmM 10 mM 25 mM 50 mM
Corr. nrnol 1 4.05 4.41 6.52 8.43 Sia/106 cells2 2 4.16 3.49 2.59 3.93
Total Acid 1 37.5 35.6 33.6 32.9 P-ase3 2 35.6 28.8 13 .1 15.1
**************************************************************
nrnol Sia 1 Unit Acid P-ase 2
0.108 0.117
0.124 0.121
0.194 0.198
0.256 0.260
13-day cells, grown in DMEM/HS/PS with the indicated [ManNAc].
2Corrected for aliquot removal and lysosomal rupture, as measured by acid phosphatase activity distribution. Analytical procedures were performed, as described in Materials and Methods.
3nrnol 4-MU released from 1 mM 4-MU-phosphate per hour per 106 cells.
135
Figure 3-6. WB6 Lysosomal Accumulation of Sia. Cells were grown in
vitro for three days. The nmole Sia/unit acid P-ase (Table 3-15) are
shown in graphic form.
Q) (I) ca • a. 'C ·u ca
:t:: s:: :::s L-Q) c. :2 o ca o
.! (I)
o E s::
WB6 Lysosomal Sialic Acid Accumulation 0.3 ""1'.------------_--.
0.2
0.1 -tT-'-'---r"--'-"r--"'---"'--'r---r-"""T"""---r~ o 10 20 30 40 50 60
ManNAc Conc., mM
-Ill- Expt.1 ..... Expt.2
137
grown in the absence of ManNAc. Figure 3-6 indicates that in WB6 cells
there was a dependence of Sia accumulation on ManNAc concentration in
the growth medium.
III.E.3. Cellular Distribution of Radioactivity Using [6-3HjManNAc
The results in Table 3-14 indicated that WB6 cells grown in 50 roM
ManNAc showed a greater increase in nmol Sia/unit acid P-ase, relative
to untreated cells, on day 2 rather than day 3. Therefore, cells were
cultured with 5JUCi/mL [6-3Hj-ManNAc in DMEM/HS/PS for two days, then
fractionated according to Figure 2-3. The radioactive molecule was used
to address some questions raised by the studies with cold ManNAc. The
main advantage to the use of [6-3Hj-ManNAc is the increased
sensitivity. The thiobarbituric acid assay requires a minimum of 2 nmol
Sia. The [6-3Hj-ManNAc available from New England Nuclear had a
specific activity of 30 Ci/mmol, and was detectable in the pmol range.
This also means that the ManNAc concentration in the growth medium can
be much lower, eliminating the perturbation of normal physiological
conditions, caused by 50 roM cold ManNAc. The radiolabelled molecule
also distinguishes newly synthesized Sia from the endogenous pool of
Sia.
Fractions were assayed for ~ -galactosidase activity to account
for lysosomal rupture. Following scintillation counting, total CPM in
a given sample was calculated. The total CPM in the lysosomal fractions
was adjusted for lysosomal rupture:
corrected Lysosomal CPM
=
138
Total Lysosomal CPM Fraction Intact Lysosomes
In these experiments, the fraction of intact lysosomes was determined
by acid phosphatase activity distribution. Cytoplasmic fractions were
corrected for lysosomal rupture by subtracting the difference between
Corrected Lysosomal CPM and Total Lysosomal CPM. Finally, both
lysosomal and cytoplasmic values were adjusted for 109 cells, to
facilitate comparisons between EL4 and WB6. The results of two
experiments are shown in Table 3-16 and Figure 3-7. These results
indicated that in EL4, 57% of the radioactivity was in the lysosomes
and 43% was in the cytoplasm. In contrast, 88% of the radioactivity in
WB6 was lysosomal, and only 12% was cytoplasmic.
The lysosomal and cytoplasmic fractions were then separated into
glycoproteins, neutral sugars, and free sialic acids, as described in
section II.G.2. These data are shown in Table 3-17. The sum of
lysosomal or cytoplasmic fractions should equal the total values shown
in Table 3-16, but this was not always the case. The percent recovery
is shown in parentheses in Table 3-17, to identify any discrepancies in
numbers. The individual values in Table 3-17 were converted to a
percentage of total CPM, where Total CPM = Lysosomal CPM + Cytoplasmic
CPM, from Table 3-16. The percent distributions are shown in Table 3-18
and Figures 3-8 and 3-9.
Tables 3-17 and 3-18 indicate that EL4 and WB6 differed in the
distribution of radioactivity within the lysosomal or cytoplasmic
139
Table 3-16. Distribution of Radioactivity in Lysosomal and Cytoplasmic Fractions of EL4 and WB6.
EL4 WB6
Expt. # Sample CPM x 10-6 % CPM x 10-6 %
1 Lysosomes 1.52 60 1.71 86
Cytoplasm 1.01 40 0.269 14
Total 2.53 100 1.98 100
2 Lysosomes 2.66 54 3.86 91
Cytoplasm 2.25 46 0.363 9
Total 4.91 100 4.22 100
Cells were cultured for two days in DMEM/HS/PS containing 5 ~Ci/mL [6-3H]-ManNAc. Lysosomes and cytoplasm were separated, as in Figure 2-3. Aliquots were assayed for 3H content using scintillation counting. CPM values were normalized for 109 cells and corrected for lysosomal rupture, as measured by acid P-ase distribution (see text for explanation). The values are also shown as percent of total CPM.
140
Figure 3-7. Cellular Distribution of Radioactivity in EL4 and WB6. The
percent CPM distribution (Table 3-16) for the two experiments were
averaged for each cell line and cell fraction.
43%
57%
Cellular Distribution of CPM in EL4
12%
Cellular Distribution of CPM in WB6
(3 Lysosomes m Cytoplasm
o Lysosomes III Cytoplasm
142
Table 3-17. Distribution of Total CPM in Lysosomal and Cytoplasmic
Fractions. The lysosomal and cytoplasmic fractions used in the
experiments in Table 3-16 were separated into glycoproteins, neutral
sugars and sialic acids, as described in Materials and Methods.
Samples were assayed for radioactivity and ~-galactosidase activity.
Values shown are CPM, corrected for lysosomal rupture and normalized
for 109 cells. The totals for each column are the sum of the
preceeding three rows. Values in parentheses are percent recoveries
of lysosomes and of cytoplasm. See text for full explanation.
143
Table 3-17. Distribution of Total CPM in Lysosomal and Cytoplasmic Fractions.
CPM x 10-5
Sample EL4 WB6
Experiment # 1 2 1 2
L:£sosomes
Lysosomal 7.76 13.0 6.08 12.5 Glycoproteins
Lysosomal Neutral 1.04 0.888 1.60 3.01 Sugars
Lysosomal Free 6.44 10.6 9.08 19.0 Sialic acids
Lysosomal Total 15.2 24.5 16.8 34.5
(100%) (92%) (98%) (89%)
C:£toplasm .
Cytoplasmic 1.93 6.53 0.0571 0.582 Glycoproteins
Cytoplasmic Neutral 7.43 13.3 2.64 2.86 Sugars
Cytoplasmic Free 0.825 2.63 0 0.377 Sialic Acids
Cytoplasmic Total 10.2 22.5 2.70 3.82
(101%) (100%) (100%) (105%)
144
Table 3-18. Percent Distribution of Radioactivity in Cytoplasmic and Lysosomal Fractions. 1
% CPM in Each Fraction
Sample EL4 WBG EL4/WBG
Experiment # 1 2 Ave. 1 2 Ave.
Lysosomes
Lysosomal 31 26 28.5 31 30 30.5 0.934 Glycoproteins
Lysosomal 4 2 3 8 7 8 0.4 Neutral Sugars
Lysosomal Free 25 22 23.5 46 45 45.5 0.516 Sialic Acids
Lysosomal Total 60 50 55.0 85 82 83.5 0.659
Cytoplasm
Cytoplasmic 8 13 11 0.3 1 0.7 16 Glycoproteins
. Cytoplasmic 29 27 28.0 13 7 10.0 2.8 Neutral Sugars
Cytoplasmic Free 3 5 4 0 1 0.5 8 Sialic Acids
Cytoplasmic 40 45 42.5 13.3 9 11.2 3.79 Total
1Numbers are the percent of total CPM in each fraction,
calculated from the data in Table 3-17.
145
Figure 3-8. Percent Distribution of Radioactivity in EL4 and WB6. The
percent values shown in Table 3-18 are illustrated in Figure 3-8. The
percent values for each cell line are shown separately. Panel a: EL4
distribution. Panel b: WB6 distribution. Abbreviation: GP,
glycoprotein.
---------------------
4.1%
29.3 %
28.7 % E;) Lysosomal GP Fa Lyso.- Neutral II Lyso. Free Sia m Cytoplasmic GP 0 Cyto. - Neutral &I Cyto. Free Sia
Total Distribution of CPM in EL4
10.6 % 0.5 %
32.2 %
~ Lyso. GP 1m Lyso. Neutral II Lyso. Free Sia m Cyto. GP 0 Cyto. Neutral II Cyto. Free Sia
48.1 %
Total Distribution of CPM in WBS
147
Figure 3-9. Percent Distribution of Radioactivity in EL4 and WB6. The
percent values shown in Table 3-18 are illustrated in Figure 3-9. The
percent values for a given fraction in each cell line are indicated in
Table 3-18.
Fraction numbers: 1) Lysosomal glycoproteins 2) Lysosomal neutral sugars 3) Lysosomal free sialic acids 4) Cytoplasmic glycoproteins 5) Cytoplasmic neutral sugars 6) Cytoplasmic free sialic acids
Total Cell ul ar 01 strl but ion of Radl oact 1 vi ty
149
fraction. This is clearer in Table 3-18, where radioactivity is
expressed as a percent of total CPM. The percentage of lysosomal
glycoproteins in EL4 and WB6 was approximately the same (EL4/WB6 =
0.934), but WB6 had much higher percentages of lysosomal neutral sugars
and free Sia (EL4/WB6 = 0.4 and 0.516, respectively). The distribution
of cytoplasmic CPM also varied between EL4 and WB6. The ratio of EL4
percent cytoplasmic glycoproteins to that in WB6 was 16. The percent
cytoplasmic neutral sugars and free Sia was also greater in EL4
(EL4/WB6 = 2.8 and 8, respectively).
III.E.4. Lysosomal stability as a Function of Time
In order to determine the velocity of lysosomal release of Sia, it
was necessary to determine the stability of EL4 and WB6 lysosomes over
the time course of the experiment. Lysosome-enriched fractions were
prepared according to Figure 2-3. The combined 480 X gav supernatants
were thoroughly mixed and divided into 5-6 tubes, one for each time
point. The samples were centrifuged at 30,000 X gav' and the
supernatants were discarded. Each pellet was gently resuspended in 1 mL
LIB (prepared at 370 C and adjusted to pH 7.4) and incubated at 370 C for
the desired time period. Following incubation, the samples were
centrifuged at 30,000 X gav. The resulting pellet and supernatant were
assayed for ~-galactosidase activity. These values were used to
calculate the percent intact lysosomes.
The results of this experiment are shown in Figure 3-10. This
figure indicates that lysosomal stability varied between time points at
-------_._--_._._----
150
Figure 3-10. Lysosomal stability at 37oC. Lysosomes were purified, as
shown in Figure 2-3. Aliquots were incubated at 370 C in LIB adjusted
to pH 7.4 at 37oC. Samples were centrifuged at 30,000 X gav' and the
resulting pellet and supernatant were assayed for ~-galactosidase
activity. Percent intact lysosomes was calculated as follows:
% Intact Lysosomes
Total Activity in 30,000 X 9 pellet Total Act. in 30,000 X g (pellet + sup.)
U) Q)
E 0 U) 0 U) ~ ..J -() m ... c --c Q) () ... Q) D.
Lysosomal Stability at 37 Degrees 100---------------------------,
80
60~ A h /\ 40
20 I ' iii ii' I iii Iii • I iii I ii' I I o 1020 3040 5060 7080 90100110120
Minutes at 37 Degrees C
I: EL41 ..... WBB
152
37oC, underscoring the need to determine the percent intact lysosomes
for each sample in an experiment. In addition, EL4 lysosomes were
somewhat more stable than those of WB6, as the average percentage of
intact EL4 lyaosomes was approximately twice as high as that of WB6,
except at zero time.
III.E.S. Velocity of Lysosomal Sialic Acid Release
Cells were grown for two days in DMEM containing S}lCi/mL [6- 3H]
ManNAc. Lysosomes were purified, according to Figure 2-3, and were
incubated, as described in section III.E.4. Aliquots of the 30,000 X
gav pellet and supernatant were assayed for ~-galactosidase activity
and 3H content, as described in Materials and Methods. Corrections of
CPM for lysosomal rupture were performed, as described in section
IILE. 3.
The corrected CPM in the supernatant was divided by the sum of
(% lysosomal free Sia CPM + % lysosomal neutral sugar CPM; Table 3-18),
in order to express supernatant CPM as a percentage of releasable CPM.
In addition, the analysis of these results accounts for the 30 minute
centrifugation (Section II.G.3) following incubation at 37oC.
Extrapolation to t = -30 minutes was performed, because it was not
possible to determine the CPM distribution without this centrifugation
step.
The results of two separate experiments are shown in Table 3-19
and Figure 3-11. The sum of the pellet CPM and the supernatant CPM
should be the same for each time point in a given cell line, but Table
153
Table 3-19. Velocity of Sia Release1 .
EL4 CPM3
Pellet Sup.
% CPM in
Sup.
% of Sol. CPM4
WB6 CPM3 % CPM in
Pellet Sup. Sup.
% of Sol. CPM4
o o
10
20
30
40 40
60
75
90
100
120
3.63 3.86
2.27
3.15
2.34
3.72 1.98
3.32
1.54
4.91
0.986
1.81
0.780 0.960
1.04
4.67
1.87
1.46 2.14
4.10
1.88
4.29
1.06
2.10
18 21
31
60
44
28 52
55
55
47
52
54
38 44
70
130
92
58 110
110
110
98
110
110
4.76 7.65
5.37
3.85
5.53
3.42 6.17
3.14
6.31
2.52
4.74
2.32
0.180 0.120
0.300
0.480
0.650
0.890 0.850
1.15
1.56
1.56
1.03
0.950
4 2
5
11
11
21 12
27
20
38
18
29
6 3
8
17
17
33 19
43
32
60
29
46
1Lysosomes were purified, as shown in Figure 2-3. Aliquots were incubated at 370 C in LIB adjusted to pH 7.4 at 370 C. Samples were centrifuged at 30,»00 X gav' and the resulting pellet and supernatant were assayed for ~-galactosidase activity and (3H) content. CPM values were corrected for lysosomal rupture, as described in the text. % in supernatant was calculated by dividing supernatant CPM by the sum of (pellet + supernatant) for a given time point, and multiplpying by 100. Two separate experiments were performed, and the data was compiled.
2minutes at 370 C.
4The percent CPM in the supernatant was divided by the sum of percent lysosomal free Sia and lysosomal neutral sugars (Table 3-18 and Figure 3-8), and multiplied by 100, to correct for different percentages of soluble CPM in the two cell lines.
-----------------------
154
Figure 3-11. Release of Lysosomal Sia. Graphical representation of the
data in Table 3-19. The y-axis is % of soluble CPM in the supernatant.
Extrapolation to t = -30 minutes was done to account for the 30,000 X
gav centrifugation time.
Velocity of Sialic Acid Release
140
i EI
120
:E ~ III III III III III
D. 100 0 CD
80~ ;/ -,g § :::J - 60 A A WBS 0 III tJ) .... 40 0 ~ 0
20
0 -40 -20 0 20 40 60 80 100 120
Minutes at 37 Degrees
156
3-19 shows that this was not the case. The simplest explanation for
this was the uneven distribution of the 480 X gav supernatant (Figure
2-3) into 5-6 aliquots.
Figure 3-11 shows that, after 40 minutes, all of the releasable
CPM in EL4 was in the 30,000 X gav supernatant. In contrast, WB6
lysosomes had not released all of their diffusible radioactivity after
two hours. Extrapolation to 100 % release in WB6 shows that these cells
would require more than 4 hours to reach 100 % CPM in the 30,000 X gav
supernatant. These results are reflected in the slope of the lines in
Figure 3-11. In EL4, the slope of the line from t = -30 minutes to t =
40 minutes was 1.34 percent/minute. In WB6, the slope of the line
through all time points was 0.359 percent/minute. The ratio of these
two slopes was 3.7, and represents the velocity of lysosomal Sia
release in EL4, relative to WB6.
III.E.6. Lysosomal Cystine Accumulation
The possibility of a generalized lysosomal transport defect in WB6
was investigated in cells incubated with 1.0 roM CDME, as described in
Materials and Methods. Aliquots of lysosomal and cytoplasmic fractions
were assayed for ~ -galactosidase activity to correct for lysosomal
rupture. The lysosomal samples were then subjected to hypotonic rupture
and centrifugation at 15,000 X gav. The supernatant was passed over a
gel filtration column to remove protein. The non-protein fractions
were pooled, lyophilized, and analyzed for cystine content. The results
of these experiments are shown in Table 3-20. The average ratio for
157
Table 3-20. Lysosomal Cystine Accumulation.
L sosomal nmol C stine x 10-2 Unit -Galactosidase Activit
EL4 Expt. Sample EL4 WB6 WB6
1 Untreated 6.64 6.76 0.982
+ 1.0 roM CDME 8.06 8.11 0.994
Ratio, +/- 1.21 1.20 1.01
2 Untreated 6.82 6.92 0.986
+ 1.0 roM CDME 6.93 7.90 0.877
Ratio, +/- 1.02 1.14 0.895
Values are lysosomal nmoles cystine (normalized for 109 cells and corrected for lysosomal rupture, as described in the text) divided by total ~-galactosidase activity in the LIB-prepared cell lysate (normalized for 109 cells).
158
lysosomal accumulation of cystine per unit ~-galactosidase in the
presence versus the absence of CDME was 1.12 ~ 0.13 for EL4 and 1.17 +
0.04 for WB6. There was no measurable difference in lysosomal cystine
accumulation in the two cell lines grown in the presence or absence of
1.0 roM CDME.
---------------- ----------
Chapter Four
Discussion
IV.A. Summary
This research has demonstrated that EL4 and WB6 had different
patterns of Sia metabolism. This conclusion is supported by several
lines of evidence. 1) EL4 and WB6 had slightly different levels of
three lysosomal glycosidases: neuraminidase, IS-galactosidase, and
~-mannosidase (Tables 3-4, 3-5, 3-6, 3-8, and 3-9). 2) WB6 cells
accumulated more lysosomal Sia than did EL4 when grown in the presence
of 50 roM ManNAc (Tables 3-13 and 3-14), and the accumulation was
dependent on the ManNAc concentration in the growth medium (Table 3-15
and Figure 3-6). The same accumulati?n occurred during incubation with
[6-3Hj-ManNAc under more physiological concentrations. 3) The
distribution of radioactive molecules (glycoproteins, free Sia, and
neutral sugars) varied in EL4 when the cells were grown in the presence
of 5)UCi/mL [6-3Hj-ManNAc. 4) The accumulation of lysosomal free Sia in
WB6 was caused, in part, by a reduced velocity of lysosomal Sia egress
in these cells. 5) There was no apparent lysosomal accumulation of
cystine, suggesting that WBG does not have a generalized lysosomal
transport defect. The last three results, presented in Tables 3-16
159
160
through 3-20, and Figures 3-7 through 3-11, will be examined in detail,
in order to support the hypothesis stated above. The remaining data
presented here, as well as earlier observations from this laboratory,
will be reviewed and interpreted in light of our proposed model for
altered sialoglycoconjugate metabolism in WB6 cells, in comparison to
EL4 cells.
IV.B. Lysosomal Transport Studies
IV.B.1. Cold ManNAc Experiments
Altered lysosomal transport of Sia is responsible for SASD
(Renlund et al., 1986a and 1986b; Mendla et al., 1988), and we propose
a similar defect for WB6 cells. Experiments using cold ManNAc (Table 3-
13) indicate that the EL4 and WB6 cells differed in the amount of
lysosomal Sia per unit acid P-ase. We chose to express the Sia content
relative to the activity of acid P-ase, rather than of a glycosidase,
as is typically done (Renl~nd et al., 1986a and 1986b; Mendla et al.,
1988), because we had observed differences in glycosidase activities in
the EL4 and WB6 (Tables 3-4, 3-5, 3-6, 3-8, and 3-9). When the cells
were grown in vitro for two days, there was essentially no difference
in the nmol Sia/unit acid P-ase in EL4 cells grown in the presence or
absence of 50 roM ManNAc (Tables 3-13 and 3-14). In contrast, the nmol
Sia/unit acid P-ase was 3.S8-fold higher in WB6, in the presence of 50
mM ManNAc, relative to untreated cells. When cells were grown for
three days in vitro, the ratio of nmol Sia/unit acid P-ase in ManNAc
161
treated cells to that in untreated cells was 1.20 for EL4 and 2.74 for
WB6 (Tables 3-13 and 3-14).
These ratios are consistent with a decreased transport of
lysosomal Sia into the cytoplasm in WB6 cells. Table 4-1 shows that
two days after dilution in medium without added ManNAc, WB6 cells had
1.78 times more endogenous lysosomal rumol Siajunit acid P-ase than did
EL4. When the cells were grown in the presence of 50 roM ManNAc, the
ratio of WB6 to EL4 rumol Siajunit acid P-ase increased to 6.33. The
same ratio three days after dilution in medium containing 50 roM ManNAc
was 2.02, and the ratio of endogenous WB6 rumol Siajunit acid P-ase to
that in EL4 was 0.888.
In our next set of experiments, we determined that Sia
accumulation was dependent on the concentration of ManNAc in the growth
medium. These experiments were performed only on WB6, as EL4 cells had
very low levels of Sia accumulation. The results shown in Table 3-15
and Figure 3-6 indicated a dependence of Sia accumulation on ManNAc
concentration in WB6 cells. The dependence did not appear to be
perfectly linear. In growth medium containing 10 roM ManNAc, there was
little accumulation relative to untreated cells. The accumulation
began between the 10 roM and 25 roM ManNAc concentrations. However, the
lower concentrations yielded quantities of lysosomal Sia that were very
close to the detection limits of the thiobarbituric acid assay (Warren,
1959). The differences may not have been detectable when working with
only 1-2 rumoles. The possibility that the ManNAc concentration affected
its uptake was later ruled out in studies with [6-3H]-ManNAc, in which
162
Table 4-1. Comparison of EL4 and WB6 Lysosomal Free Sia/Unit Acid P-ase in the Presence or Absence of 50 roM ManNAc1 .
Days of In WB6 Untreated WB6 + 50 roM ManNAc Vitro Growth EL4 Untreated EL4 + 50 roM ManNAc
2 1. 78 6.33
3 .888 2.02
Ratio, Day 2 2.00 3.13 Day 3
1Numbers are the ratio of nmole Sia/unit acid P-ase in cells grown in the presence or the absence of 50 roM ManNAc (Table 3-13).
163
EL4 and WB6 incorporated similar amount of radioactivity when the
ManNAc concentration was 0.17;aM (Table 3-17).
Several alternative explanations for the lysosomal Sia
accumulation in vffi6 cells can be ruled out by experiments with
[6-3H]-ManNAc, and by neuraminidase stUdies. Table 3-16 shows that EL4
and WB6 incorporated similar amounts of ManNAc, even at very low
concentrations, implying that variations in ManNAc uptake were unlikely
to be responsible for the observed differences in lysosomal Sia
accumulation (Table 3-15 and Figure 3-6). The accumulation of free Sia
in WB6 was not due to increased entry of sialoglycoconjugates into the
lysosomes, as lysosomal glycoprotein levels were similar in the two
cell lines (Table 3-18 and Figures 3-8 and 3-9). Release of Sia from
glycoproteins by neuraminidase was similar in EL4 and WB6 (Tables 3-4
and 3-8), suggesting that the accumulation was not a result of enzyme
differences in EL4 and WB6.
The results of the cold ManNAc experiments must be interpreted
with caution, for several reasons. The ManNAc concentration in the
growth medium far exceeded physiological concentrations, although
others have used ManNAc concentrations as high as 100 roM (Renlund et
al., 1986a). This concentration was necessary in order for the cells to
metabolize ManNAc to Sia in quantities high enough to be detected in
the thiobarbituric acid assay (Warren, 1959). ManNAc is derived from
the epimerization of carbon 2 of UDP-GlcNAc (Roehrig, 1984). The
GlcNAc, in turn, is synthesized from Glc by way of fructose-6-
phosphate. The DMEM used to culture EL4 and WB6 contains 1000 mg/L Glc,
-----------------------
164
which is equivalent to 5.6 roM. Only a small fraction of this is used
for Sia biosynthesis. The addition of 50 roM ManNAc, therefore, greatly
increases the supply of Sia precursors, and may alter the normal
distribution of sialoglycoconjugates.
There are other disadvantages to the use of high concentrations of
cold ManNAc to study the biosynthesis of Sia in EL4 and WB6.
Differences in the uptake of ManNAc into the cytoplasm could not be
determined, as there are no specific biochemical assays for ManNAc
which had a high enough sensitivity. Another disadvantage is that the
Sia found in the lysosomes was not necessarily synthesized from
exogenously added ManNAc. The distribution of sialylated molecules
other than free Sia could also not be determined, due to the detection
limits of the thiobarbituric acid assay (Warren, 1959).
IV.B.2. 3H-ManNAc Studies
The encouraging results with 50 roM cold ManNAc convinced us to
attempt the experiments with [6-3H]-ManNAc. Table 3-16 shows that EL4
and WB6 incorporated similar amounts of [6-3H]-ManNAc, because the
ratios of (EL4 Total CPM)/(WB6 Total CPM) in experiments one and two
were 1.28 and 1.16, respectively. The data in Table 3-16 confirm that
WB6 accumulated more radioactivity in the lysosomes than did EL4. On
average, 57% of the radioactivity in EL4 was lysosomal, and 43% was
cytoplasmic. In contrast, the average lysosomal and cytoplasmic
percentages in WB6 were 88% and 12%, respectively.
Additional experiments were performed on the lysosomal and
165
cytoplasmic fractions used in Table 3-16 and Figure 3-7, in order to
determine the chemical form of the radioactivity. Ammonium sulfate
precipitation and anion exchange chromatography enabled us to separate
each fraction into glycoproteins, neutral sugars, and free Sia. These
results are shown in Tables 3-17 and 3-18, and Figures 3-8 and 3-9.
The differences in distribution of radioactivity in EL4 and WB6 were
most clearly seen in Table 3-18, where individual CPM values were
converted to a percentage of total CPM. The accumulation of Sia in the
lysosomes, due to decreased transport of Sia into the cytoplasm, may
have affected the enzymes involved in Sia metabolism (Figure 1-6). The
differences in cellular distribution of radioactivity (Table 3-16 and
Figure 3-7) were probably a consequence of variations in activity of
one or more of these enzymes (Figure 1-6), since the [6-3H]-ManNAc
entered the cell to the same degree in both cell lines (Table 3-16).
The CPM from [6-3H]-ManNAc are shown as "Cytoplasmic Neutral
Sugars" and "Lysosomal Neutral Sugars" in Table 3-17, and the percent
of total CPM for these quantities is indicated in Table 3-18. The major
product of ManNAc metabolism is Sia, and the major product of Sia
catabolism is ManNAc (Diaz and Varki, 1985). The radioactivity that
eluted from the anion exchange columns in 10 mM NaCl was presumably
ManNAc. However, it was not unequivocally shown to be ManNAc, so the
term "neutral sugars" will be used. The percentage of cytoplasmic
neutral sugars was 2.8-fold higher in EL4 than in WB6. This suggests
that WB6 converted more of the ManNAc to Sia, or that the free Sia
exiting the lysosomes in EL4 was readily converted back to ManNAc
166
(Figure 1-11). The ratio of EL4/WB6 average percentage of lysosomal
neutral sugars was 0.4 ~ 0.2. The radioactivity in this fraction may
have been due to incomplete separation of lysosomes and cytoplasm, or
it may have been caused by traces of Sia that failed to bind to the
anion exchange column. These are likely possibilities, as one would
not expect to find any lysosomal neutral sugars derived from ManNAc.
These values may also be artifacts of the method used to account for
lysosomal rupture.
Lysosomal rupture was calculated on the basis of acid hydrolase
assays. After adjusting the enzyme activity in the initial cell lysate
for unbroken cells, the fraction of intact lysosomes was calculated as
follows:
Fraction Intact = Total Activity in Purified Lysosomes Lysosomes Total Activity in Cell Lysate
In initial experiments, two enzymes (acid P-ase and ~-galactosidase)
were ;::>.~sayed in a given experiment, to test the reliability of this
method for determining the fraction of intact lysosomes. This value was
the same in a given experiment, regardless of which enzyme activity was
used in its calculation. Radioactivity in lysosomal sa~ples was
divided by the fraction of intact lysosomes to correct for rupture. The
difference between this number and the uncorrected value was subtracted
from the corresponding cytoplasmic sample.
The lysosomal and cytoplasmic glycoproteins were assumed to be
sialoglycoproteins, on the basis of their 3H content. Sia is the only
product of ManNAc metabolism known to be used in glycoprotein
167
oligosaccharides (Oiaz and Varki, 1985). The two cell lines had very
similar percentages of lysosomal glycoproteins, which indicated that
the flux of sialoglycoproteins through the lysosomes was the same in
EL4 and WB6 (Table 3-18).
Unlike the lysosomal fraction, the cytoplasmic glycoprotein
content differed greatly between the two cell lines (Table 3-18). The
percent of cytoplasmic glycoproteins in EL4 was 16 times greater than
it was in WB6. Increased levels of CMP-neuraminate synthetase (enzyme 1
in Figure 1-6) and/or sialyltransferase (enzyme 2 in Figure 1-6) in EL4
could account for this finding. Alternatively, it is possible that WB6
delivers more of its sialoglycoproteins to the cell surface. However,
radioactivity associated with the PM was not determined in this
experiment. Previous observations in this laboratory, suggesting a
relationship between H-2Kb antigenic density and immunogenicity (Gilmer
et al., 1984; Flores and Gilmer, 1984) are consistent with this theory,
and will be discussed below.
The most significant data in Table 3-18 are the percentages of
free Sia. The ratio of EL4/WB6 percentage of cytoplasmic free Sia was
8 + 1. The percentage of lysosomal free Sia in EL4, relative to WB6,
was 0.52 ~ 0.04. WB6 had nearly twice as much of its total
radioactivity, relative to EL4, as free Sia (Table 3-18). This is
consistent with the model for SASO proposed by Renlund et al. (1986a),
except that they observed a 4.6-fold increase in free Sia in SASO
patients, relative to normal controls.
The velocity of lysosomal Sia transport was studied in EL4 and
168
WB6, to determine the underlying cause of the Sia accumulation in WB6.
Lysosomal stability over the time course of the experiment was
investigated in Figure 3-10. These data show that radioactivity in each
sample needed to be corrected for lysosomal rupture, as described
above. Table 3-19 and Figure 3-11 show the time course of Sia egress
from EL4 and WB6 lysosomes the cells were grown in the presence of
[6-3Hl-ManNAc at 5jUCi/mL (0.167)lM) for two days. The graph includes
a point at -30 minutes, to account for the 30,000 X av centrifugation
time. In EL4 cells, all of the releasable radioactivity was in the
supernatant within 40 minutes at 37°C. In WB6 cells, only 50% of the
radioactivity was released after 2 hours. The slopes shown in Figure 3-
11 are the velocity of Sia egress for the two cell lines. The velocity
in EL4 in the time period from -30 minutes to 40 minutes was 1.34
percent/minute. In contrast, the velocity of WB6 lysosomal Sia release
was 0.359 percent/minute over the entire time course of the experiment.
The ratio of these values is 3.7, and represents the relative velocity
of Sia egress in EL4 cells.
These data provide support for our hypothesis of altered Sia
metabolism in WB6 cells. We propose that Sia accumulates in WB6
lysosomes as a result of a decreased velocity of Sia transport to the
lysosomes. Four models could account for these findings, and all are
consistent with other results described in Chapter Three. One
possibility is that WB6 has a defective lysosomal Sia transporter. A
second is that the lysosomes in WB6 cells are larger, with a fixed
number of transporters per lysosome. Third, there could be more
169
lysosomes in WB6, with a fixed number of transporters distributed
evenly among them. Finally, there may be decreased transport of 0-
acetylated sialic acids.
We currently favor the latter three alternatives, as we have not
yet undertaken studies of the proposed Sia transporter at the protein,
RNA, or gene level. EL4 and WB6 had very different growth patterns
following dilution with fresh DMEM/HS/PS (section III.A.). Thus, it is
reasonable to postulate different numbers and/or sizes of lysosomes in
the two cell lines. Larger lysosomes in WB6 cells would imply that
they could contain more Sia. The increased sensitivity of WB6 lysosomes
to NaCl concentration in RSB (Table 3-3 and Figure 3-4) could reflect
their larger size. The increase in lysosomal membrane
phospholipid:protein ratio in WB6 (Table 3-12) on day 2, relative to
day 3, suggests that one mechanism by which WB6 responds to cell growth
is to alter lysosome size. Similarly, variations were observed in WB6
acid P-ase activity on day 2 and day 3. If acid P-ase varies as total
lysosomal volume varies, these data could be another indication of
increased lysosomal size and/or quantity in WB6, relative to EL4.
Several experiments are proposed to distinguish between variations
in lysosome size and lysosome quantity differences in EL4 and WB6.
Determination of the actual quantities of lysosomal membrane
phospholipid and protein in EL4 and WB6 would be be one method of
comparing the numbers of lysosomes in the two cell lines. Light
microscopy and/or electron microscopy of acid P-ase-stained cells
(Holtzmann, 1989) would permit quantitation of lysosomal size as well
170
as quantity. Studies of the velocity of cystine egress, rather than
just accumulation (Table 3-20), would also help distinguish between
these two possibilities. A decreased rate of cystine egress in W86
would support the proposal that W86 has larger lysosomes, if one
assumes that the number of cytine tranporters per lysosome was fixed.
Failure to demonstrate decreased velocity of cystine egress in W86
would be evidence for defective Sia transporters in these cells.
IV.C. Acid Hydrolase Studies
IV.C.1. Glycosidase Activities
This research began with the hypothesis that W86 would have more
neuraminidase activity than EL4, due to its reduced level of cell
surface Sia (Table 1-1). Studies using RSB and LIB showed that, in
general, W86 had less neuraminidase activity than EL4 (Tables 3-4 and
3-8). The range in total activity in the two cell lines did not
overlap (Table 3-4), but the experiments using RSB-prepared "lysosomes"
failed to take lysosomal rupture into account. The LIB data, however,
did take this into account, but our method of accounting for lysosomal
rupture was based on the distribution of enzyme activity. Although
this method is widely used in the literature (Renlund et al., 1986a and
1986b; Mendla et al., 1988; Smith et al., 1987), errors in the
"accounting" assay will propagate errors into a second set of results.
In short, it is questionable how significant the differences are in
neuraminidase activity in EL4 and W86.
171
EL4 had slightly greater ~ -galactosidase (Table 3-6 and Table 3-
10) and o(-mannosidase (Table 3-7) activities than WB6. The finding
that all three lysosomal glycosidase activities studied varied in the
same direction in EL4 and WB6 suggests several possible mechanisms of
co-regulation of these enzymes. A simple explanation for these results
is inhibition of neuraminidase by free Sia in WB6 lysosomes. Another
possibility is modification of the other two enzymes. At the present
time, we have no experimental evidence for these two alternatives,
although we favor the former.
The activity of ~ -galactosidase and/or 0( -mannosidase may be
affected by substrate availability and/or product accumulation. Sia is
the terminal sugar, and Gal is the penultimate sugar, on complex N
linked oligosaccharides (Figure 1-4). p-galactosidase is an
exoglycosidase, and can, therefore, only cleave Gal if Sia has been
removed by neuraminidase. Similarly, o(-mannosidase can only cleave
Man residues if Sia, Gal and GlcNAC are removed. One possible mechanism
of co-regulation is feedback inhibition of ~-galactosidase and
o(-mannosidase by the sialoglycoproteins that would accumulate when
neuraminidase activity decreases. Any decrease in neuraminidase
activity reduces the availability of substrates for the other two
glycosidases. It is possible that the unavailability of natural
substrates could affect enzyme activity, in an indirect manner.
Decreased levels of neuraminidase activity would result in the
accumulation of sialyloligosaccharides. These molecules might inhibit
~ -galactosidase and o(-mannosidase activity, even in the presence of
172
synthetic 4-MU substrates. Although we were unable to find any support
for this theory in the literature, it could easily be tested by
purifying sialyloligosaccharides from WB6 cells, and testing their
ability to inhibit EL4 #-galactosidase and/or o(-mannosidase.
An alternative explanation for the simultaneous reduction in all
three glycosidases in WB6 is regulation of ,8-galactosidase and 0(
mannosidase activity by neuraminidase. The Neu-1b locus in C57BL/6 mice
is a single gene on chromosome 17, near the H-2 complex (Womack et al.,
1981). The product of this gene, neuraminidase, affects lysosomal
processing of other acid hydrolases, including acid P-ase,
0<. -mannosidase, 0( -glucosidase, and aryl sulfatase (Klein, 1986). The
consequence of decreased neuraminidase activity is hypersialylation of
these enzymes, which may affect their activity. Although
hypersialylation of ~-galactosidase was not tested in the studies
cited by Klein (1986) it is very likely that this enzyme would be
affected by neuraminidase deficiency (Womack et al., 1981).
Since EL4 originated in C57BL/6 mice, these results are especially
relevant to our studies. The fact that Neu-1b is linked to the H-2
complex has implications :n the syngeneic immune response to these
cells, as discussed below. Isoelectric focusing of purified lysosomal
enzymes would indicate whether WB6 enzymes were hypersialylated. If so,
de-O-acetylation and neuraminidase treatment of these molecules, prior
to assay with 4-MU-Sia, would yield similar activities in EL4 and WB6,
if the decreased activity in WB6 is a result of hypersialylation.
173
IV.C.2. Acid P-ase Activity
Acid P-ase was selected for investigation because it is a
characteristic lysosomal acid hydrolase not directly involved in
carbohydrate metabolism. Our goal was to confirm or refute the finding
that the EL4/WB6 ratios of neuraminidase activity and of
~-galactosidase activity were dependent on the number of days after
dilution with fresh medium. Acid P-ase activity was dependent on the
number of days after dilution with fresh medium (Table 3-11). This is
a reflection of variations in cell density, and presumably in number of
lysosomes. Two days after dilution, the ratio of EL4 acid P-ase
activity to that in WB6 was 1.4 ~ 0.3, while on day 3 the same ratio
was 0.79 ~ 0.09 (Table 3-11).
A comparison of the EL4/WB6 ratios on day 2 and day 3 for
neuraminidase, ~-galactosidase, and acid P-ase is shown in Table 4-2.
The average EL4/WB6 ratio for all three enzymes was 1.5 ~ 0.2 on day 2,
and was 0.84 ~ 0.06 on day 3. If one assumes that the activity per
lysosome does not change, then these data suggest that the relative
number of lysosomes changed from day 2 to day 3. Typically, EL4 cells
grew to higher density and viability on day 2, relative to WB6. On day
3, the reverse was true. WB6 frequently grew to densities greater than
1 X 106 cells/mL on day 3, whereas the maximum growth density of EL4
was usually less than 8 X 105 cells/mL by day 3. The variations in
amount of lysosomal activity during cell growth must be accounted for
in subsequent experiments involving lysosomal metabolism, as discussed
below.
174
Table 4-2. Comparison of EL4/WB6 Activity Ratio for Three Lysosomal Enzymes 1.
Number of Days of In Vitro Growth
Enzyme Two Three
Neuraminidase 1. 76 o.sos
-Galactosidase 1.37 0.932
Acid P-ase 1.4 0.79
Average 1.5 + 0.2 0.S4 + 0.06
1Nurnbers represent the ratio of total enzyme activity in EL4 relative to WB6,in LIB-prepared cells. Neuraminidase data were taken from Table 3-S, ~-galactosidase data were taken from Table 3-9, and acid P-ase data were taken from Table 3-11.
175
IV.C.3. Acetylesterase Activity
The results of the acetylesterase assays (Table 3-10) are exactly
as we would predict, based on the data in Table 1-2 and the metabolic
pathways shown in Figure 1-6. EL4 had a lower proportion of 9-0Ac-S
NAcNeu, relative to WB6, and this is consistent with elevated
O-acetylesterase activity. Table 3-11 shows that EL4 had 1.8-fold more
acetylesterase activity than WB6 after three days of in vitro growth.
This was in contrast to the three acid hydrolases shown in Table 4-2,
where the average EL4/WB6 activity ratio on day 3 was 0.84.
The substrate used in these experiments was 4-MU-Ac. The esterases
which hydrolase this substrate are not necessarily specific for 0-
acetylsialic acids. However, Varki and Diaz (1986) identified a human
erythrocyte esterase which is specific for O-acetylsialic acids. They
also showed that this esterase is identical to esterase D. The latter
was first identified by its ability to cleave 4-MU-Ac, as this ester is
a poor substrate for most RBC esterases (Scott and Wright, 1978).
Ideally, O-acetylsialic acids should be used as substrates for
determination of O-acetylsialic acid esterase activity. As mentioned
previously, O-acetylsialic acids are unstable and extremely difficult
to purify (Varki and Diaz, 1984). Purification and characterization of
these molecules is currently underway in this laboratory, and the 0-
acetylesterase ~ill be re-examined when O-acetylsialic acid substrates
become available.
176
IV.D. Lysosomal Membrane Phospholipid and Protein Studies
The acid P-ase studies (Table 3-11) suggested that there might be
an alteration in the numbers of lysosomes during the growth cycle of
EL4 and WB6. The number of lysosomes appeared to decrease in EL4
between day two and day three, and it appeared to increase in WB6.
When the experiments in Table 3-12 were performed, we were mainly
concerned with the phospholipid:protein ratio. Our reasoning was that a
constant lysosomal phospholipid:protein ratio would indicate that the
amount of acid P-ase per lysosome was unchanged. We found that, two
days after dilution, EL4 and WB6 had very similar ratios of lysosomal
membrane phospholipid:protein. However, three days after dilution, the
phospholipid:protein ratio had increased by 15% in WB6, but was
essentially unaltered in EL4 (Table 3-12). This resulted in a decrease
in the phospholipid:protein ratio of EL4/WB6, from 1.02 ~ 0.06 on day
2, to 0.866 on day 3.
It was not possible to calculate the absolute amount of
phospholipid and protein, as the samples were not corrected for
lysosomal rupture. If the number of lysosomes in WB6 is dependent on
the metabolic state of the cells, as the data in Table 3-12 suggest, it
would be useful to repeat the phospholipid and protein analysis to
calculate the absolute quantities as well as the ratio.
--------------------- - --------------
177
IV.E. A Model for Altered Sialoglycoconjugate Metabolism in WB6 Cells
These results support our hypothesis EL4 and WB6 have altered
patterns of Sia metabolism. The velocity of lysosomal Sia release in
WB6 cells was 3.7-fold less than the same quantity in EL4. The
decreased velocity in WB6 is most likely due to larger lysosomes in
WB6. This results in the accumulation of free Sia inside the lysosomes.
We propose that there are several consequences of this accumUlation,
all of which are consistent with earlier observations from this
laboratory. Considerable work remains to be done identify cause-and-
effect relationships between the lysosomal metabolism of Sia and the
cell-surface properties of EL4 and WB6 reported previously (Gilmer et
al., 1982; Gilmer et al., 1984; Flores and Gilmer, 1984).
The increased level of 9-0Ac-S-NAcNeu on the surface of WB6 cells
(Table 1-2), in comparison to EL4, may result from the increase in
lysosomal free Sia. This accumUlation might be a signal to reduce the
flux of sialoglycoproteins through the lysosomes, as degradation of
these molecules would further increase the lysosomal Sia content. The
data in Figure 3-19 suggest that there is no difference in the level of
lysosomal glycoproteins after two days of in vitro growth. However,
these data do not prove or disprove the hypothesis that the time of
passage through the lysosomes varied in EL4 and WB6, since they are
based on a static picture of the level of lysosomal sialoglycoprotein.
The cytoplasmic sialoglycoproteins might instead be shuttled to the
Golgi, via endosomes, for 9-0-acetylation by a specific 0-
178
acetyltransferase (Figure 1-G). The relative amount of 9-0Ac-5-NAcNeu
on the cell surface would then increase in WBG. In addition, if
lysosomal sialoglycoprotein catabolism was inhibited in WBG, fewer
cell-surface sialoglycoproteins would be recycled through the
lysosomes. Instead, they would accumulate on the cell surface. This
was observed in the WGA-resistant variant WD1 (Table 1-1), which had
physicochemical properties similar to WBG. Although the studies have
not been done on WBG, we predict on the basis of Table 1-1 that it has
similar H-2Kb antigenic density to WD1, and higher than that of EL4.
The recycling of H-2 molecules has been studied in many systems,
including EL4. Fishman and Cook (198G) demonstrated that lysosomes are
involved in the recycling of cell-surface molecules. After
radiolabelling cell-surface sialoglycoconjugates (periodate/NaB3H4 ),
they observed radioactivity in the lysosomes in 30-60 minutes, and in
the Golgi after 3 hours. Reichner et al. (1988) found that some
molecules can be reprocessed by Golgi enzymes and return directly to
the cell surface, without passing through a lysosomal compartment.
Others have shown (Tse et al., 1984; Pernis, 1985) that this recycling
is selective for certain cell-surface molecules, including class I MHC
antigens. The glycosylation of class I molecules also appears to playa
role in the transport and cellular distribution of class I molecules.
Taken together, these results support our hypothesis of altered
sialoglycoprotein recycling in WB6, as a result of lysosomal Sia
accumulation.
179
Increases in the cell-surface antigenic density may be responsible
for the immunogenicity of WB6. Like W01, these cells are unable to grow
in vivo. Instead, many small cells, presumably CTL, are generated in
response to syngeneic stimulation with WB6 (Tinsley and Gilmer, 1987).
The syngeneic response to WB6 is directed against unique tumor antigens
in association with its class I H-2 molecules, one of w' ~ch is H-2Kb •
Previous studies have shown that the cell-surface den~ity of H-2
molecules plays an important role in cell-mediated lysis (Goldstein and
Mescher, 1987; Shimonkevitz et al., 1985). A similar mechanism could be
operating in our system, resulting in the different syngeneic response
to EL4 and WB6 (Table 1-1). The H-2Kb antigenic density in EL4 could be
below the threshold identified by Goldstein et ale (1987). With too
few H-2 molecules with which to associate, the tumor antigens may not
be recognized by the animal's immune system. When the antigenic density
exceeds this minimum, as we propose for WB6, CTL recognition and lysis
could occur.
An alternative explanation for the increased CTL recognition and
lysis of WB6 lies in the O-acetyl modification. The presence of
increased amounts of cell-surface 9-0Ac-5-NAcNeu could be a recognition
signal for CTL. The CTL recognition domain may include carbohydrate
(Pimlott and Miller, 1984, 1986). Just as 9-0-acetylation of Sia
affects WGA binding (Maget-Dana et al., 1981), the same modification
may affect CTL recognition. The syngeneic response to WB6 is probably
directed against processed tumor antigens in association with H-2. The
O-acetyl moiety may somehow enhance this association, making it more
180
recognizable to the immune system. Further study of the "presumed" CTL
may help address this question. If the O-acetyl moiety forms part of a
CTL recognition domain, the pre-treatment of WBG target cells with Sia
O-acetylesterase should inhibit recognition and lysis. However, the
cells may be able to replace the O-acetyl moiety using endogenous 9-0-
acetyltranferase. A more definitive experiment would be to select cells
naturally high in 9-0Ac-5-NAcNeu using IVC in which the esterase was
inactivated (Muchmore and Varki, 1987; Varki, A., personal
communication, 1989). Addition of fluorescent antibodies to IVC would
permit fluorescence-activated cell-sorting of cloned cells. Cells
selected in this manner could then be used as targets in a CML assay,
in which the effectors were the anti-WBG "CTL".
The immunogenicity of WBG may be related to the genetic linkage
between the ~ locus and the neuraminidase Neu-1 gene, as discussed
earlier (Womack et al., 1981). The product of this gene may affect the
sialylation of H-2 molecules. Reduced levels of neuraminidase, as
observed in WBG cells, would mean that' at any given time, a greater
fraction of molecules were sialylated. This would result in a larger
pool of potential substrates for 9-0Ac-5-NAcNeu transferase, and
ultimately lead to increased levels of H-2 molecules containing 9-0Ac-
5-NAcNeu.
These studies have identified a fundamental biochemical defect in
the WGA-resistant cell line WBG. It is likely that this is the primary
defect, as most other observations from this laboratory can be
explained with this model. However, this research has not identified a
181
direct cause-and-effect relationship between lysosomal Sia transport,
acid hydrolase activities, O-acetylation of Sia, and/or immunogenicity
of WBG cells.
Future directions in this research, other than single experiments
mentioned above, should include protein and genetic analysis of EL4 and
WBG to identify a potential Sia transporter in the lysosomal membrane.
Any observed protein differences may be the result of an altered gene
for the transporter, or from altered transcription and/or translation
of this molecule. Although the data presented in Chapter 3 suggest that
WB6 differs from SASD cells in several important aspects,
identification of a Sia transport system in WBG may ultimately aid in
our understanding of the Sia transport defect responsible for the human
disorder SASD. The lysosomal transport of Sia may also have
implications in the treatment of cancer, if we are able to demonstrate
a relationship between lysosomal Sia accumulation and the clearance of
these cells by the immune system.
--------------
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