β-galactosidase from rat epididymal fluid is bound by a recognition site attached to membranes of...

Vol. 143, No. 3, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

March 30, 1987 Pages 799-807

P-GALACTOSIDASE FROM RAT EPIDIDYMAL FLUID IS BOUND BY A

RECOGNITION SITE ATTACHED TO MEMBRANES OF THE EPIDIDYMIS

DIFFERENT FROM THE PHOSPHOMANNOSYL RECEPTOR

Miguel A. Sosa, Luis S. Mayorga, and Francisco Bertini

Instituto de Histologia y Embriologia, Facultad de Ciencias Medicas,

Universidad National de Cuyo, Casilla de correo 56, Mendoza 5500

Argentina

Received December 31, 1986

SUMMARY: In order to know if the P-galactosidase of the rat ep'riidymal fluid, as other secreted

acid hydrolases, carries a marker in its molecule, we studied the binding of this enzyme to cel-

lular membranes of the epididymal tissue. The binding, like that mediated by the phosphomannosyl

receptor, was saturable, did not require calcium, had a Kd in the nM range and was inhibited by

phosphatase or metaperiodate treatment of the enzyme. However fructose 6-phosphate derivates

were more effective competitive inhibitors than mannose 6-phosphate. The binding capacity of

the membranes were extractable with Triton X-100 and incorporable into liposomes. Trypsin

inhibited the binding capacity of Triton extracts but it did not affect the affinity of intact

cellular membranes for fl-galactosidase. The results suggest that a phosphorylated carbohydrate

of the enzyme is bound by a recognizing site of the cellular membranes different from the

phosohomannosyl receptor. 0 1987 Academic Press, Inc.

Acid hydrolases are synthesized in the rough endoplasmic reticulum and translocated to

lysosomes through the Golgi complex (2). It is now well known that receptors attached to subcel-

lular membranes, which recognize phosphomannosyl residues in the enzymatic molecule, play an

important role in this transport in various cellular types and tissues (2). The phosphomannosyl

marker is lost when the enzymes arrive to lysosomes as a consequence of the processing of the

proteins to their mature forms (5). Cultured fibroblasts secrete part of the newly synthesized

lysosomal enzymes (6). The secreted enzyme retains the recognizing marker and is bound with high

affinity by subcellular membranes containing phosphomannosyl receptors (3). Not only fibroblasts

leak acid hydrolases; the presence of these enzymes in most body fluid has been interpreted as

a consequence of the secretory activity of various types of cells (8,9). For instance, the

epididymal fluid is particularly rich in these enzymes, and its hydrolytic activity appears to

stem from the epithelial cells of the duct (4). Then, we found of interest to known if these

Abbreviations: EGTA, ethyleneglycol-bis-(fi- aminoethyletherj-N,N,N',N'-tetraacetic acid.

0006-291X/87 $1.50

799 Copyright 0 I987 by Academic Press, Inc.

All rights of reproduction in any form reserved.

Vol. 143, No. 3, 1987 BIOCHEMICAL AND EU~PHYSKAL RESEARCH COMMUNICATIONS

acid hydrolases carry a recognizing marker like the fibroblast .enzymes, and we studied the

binding of @-galactosidase of the rat epididymal fluid to subcellular membranes of the tissue.

The results show that this enzyme is bound with high affinity by the membranes, but the binding

is more sensitive to fructose 6-phosphate derivates than to mannose 6-phosphate.

MATERIALS AND METHODS

Materials - P-

Galactosidase substrates, carbohydrates, lipids and Triton X-100 were pur-

.chased from Sigma Chemical Co., Hyamine 2389 and saponin were obtained from BDH Chemical Ltd.

Enzyme Preparation - Rat epididymal fluid was obtained by perfusion of a segment of the cauda

epididymidis as previously described (11). The fluid was cooled at 4"C, diluted with 0.15 M NaCl

and the sperm cells were separated by centrifugation at 3,000 x g for 5 min. The supernatant

was applied to a DEAE cellulose column of 1 x 5 cm equilibrated with 20 mM sodium phosphate buf-

fer, pH 6 (phosphate buffer), and eluted with the same buffer. The fractions with the higest

activity (9,DOO-10,000 units / mg of proteins) were froze at -5°C. In this condition the binding

activity diminished after 3 week of storage.

Chemical and Enzymatic Treatment of fl-Galactosidase - The enzyme was incubated in phosphate

buffer containing 5 mM sodium metaperiodate and 0.15 M NaCl at 4°C for 3 h in the darkness. The

reaction was stopped by the addition of glycerol (2 M final concentration) and the mixture was

dialysed for 24 h against phosphate buffer. A 25-305 of the original enzymatic activity was re-

covered after this treatment. Phosphatase treatment of @-galactosidase was carried out accord-

ing to Ullrich et a1.(15). The enzyme was dialysed overnight against 50 mM Tris-HCl buffer, pH

7.5 containing 1 mM MgCl 2'

Alkaline phosphatase (Sigma, type III) was added to a final concen-

tration of 1.2 units/ml. The mixture was dialysed for 4 h at 4°C and for 2 h at 37°C against

the Tris-HCl buffer. In this condition less than 7% of the P-galactosidase activity was lost.

The treated enzymes were used for binding assays and compared with control enzymes which were

treated as described above, but without the addition of metaperiodate or alkaline phosphatase.

Some controls were carried out adding alkaline phosphatase to the binding assay.

Preparation of Rat Epididymal Membranes - After the perfusion of the cauda epididymidis, the

whole organ was trimmed of fat, chopped with scissors and homogenized 1:5 (w/v) in 0.25 M

sucrose, 10 n+i Tris-acetate buffer, pH 7.4, 3 mM EDTA (sucrose buffer) in a glass homogenizer

with Teflon pestle. The homogenate was centrifuged at 600 x g for 10 min at 4°C in a Sorvall

RCZ-B centrifuge using a SS-34 rotor to eliminate spermatozoa, nuclei and tissue debris. The

supernatant was centrifuged at 48,000 x g for 30 min and the sediment was sonicated for 10 set

and washed twice with the sucrose buffer containing 0.5% saponin and 0.6 M KC1 to remove soluble

proteins and the endogenous fl-galactosidase activity of the membranes (residual activity: less

than 30 units / mg of proteins). Finally, the membranes were washed and resuspended in the phos-

phate buffer and stored at -5'C. The binding activity of these membranes was stable for more

than 3 months.

Trypsin Treatment of Cellular Membranes - The membranes were incubated at 37°C for 5 min in

0.05 M Tris-HCl buffer, pH 7.8 containing 1 ti MgC12, 1 mM CaCl , 0.2 M KC1 and 1 mg/ml trypsin

(Sigma, type III); then they were washed twice with the same bt?ffer without trypsin and once with

phosphate buffer. These membranes were used for binding assays in the presence of 0.1 mg/ml

trypsin inhibitor.

Triton X-100 Extract of Membrane Proteins. Incorporation into Liposomes - Cellular membranes

were resuspended in phosphate buffer containing 0.1% Triton X-100. Thirty hours later, the mix-

ture was centrifuged at 40,000 x g for 20 min and the supernatant was dialysed overnight against

phosphate buffer and for 24 h against 0.02 M Tris-HCl buffer, pH 7.8. After the dialysis the

800


extract was incubated at 37'C for 10 min with or without the addition of 1 mg/ml trypsin.

Liposomes were prepared using 63 umoles of phosphatidylcholine, 18 umoles of dicetylphosphate

and 9 umoles of cholesterol solubilized in 5 ml of chloroform. Aliquots of 0.1 ml of this

solution were evaporated and the films were resuspended using phosphate buffer, trypsin treated

extract or untreated extract. After a 5 set sonication, the samples were dialysed overnight

against phosphate buffer. The liposomes were then centrifuged at 40,000 x g for 20 min, washed

twice with 0.2 M KC1 and once with phosphate buffer, and used for binding assay in the presence

of 0.1 mg/ml trypsin inhibitor.

Binding Assay - Membrane proteins were incubated at 20°C in 4 ml polypropylene tubes

containing @-galactosidase in 0.25 ml of phosphate buffer. After 60 min, 1.5 ml of cold

phosphate buffer were added. The tubes were then stirred in a Vortex mixer for 30 set and

centrifuged at 48,000 x g for 15 min. The pellets were washed with 2 ml of the same buffer

and their enzymatic activity was measured.

Assays - fi-Galactosidase was assayed fluorometrically (12) using 4-methylumbelliferyl-

@-D-galactopyranoside (0.8 M) in 0.13 M sodium citrate buffer, pti 4 with 0.1% Triton X-100.

One unit of activity is the amount of enzyme which catalyzes the release of 1 nmole of 4-methyl-

umbelliferone / h. Protein concentration was measured by the method of Lowry et a1.(13).

Total lipids were measured using vanillin in a sulfuric-phosphoric acid medium.

RESULTS

@-Galactosidase of the rat epididymal fluid was bound with high affinity by membranes

of this organ. The amount of bound enzyme was proportional to the amount of membrane proteins

used in the assay and it did not significantly decrease after three washes of the membranes

with phosphate buffer (data not shown). The binding, like that mediated by the phosphomannosyl

receptor (3), was saturable, had a Kd in the nM range (assuming a specific activity of 0.19

units / fmole of enzyme, similar to that reported for fl- galactosidase of human liver (1411,

did not require calcium, and was resistant to unphosphorylated carbohydrates (Fig. 1 and Table

I). tlowever, fructose 6-phosphate derivates, that do not prevent the endocytic uptake of

0 120 240 360 10 20 30

UNITS ADDED UNITS BOUND

Figure 1. Binding of o-galactosidase of rat epididymal fluid to cellular membranes of the tis-

sue. Forty ug of membrane proteins were incubated at 20°C for 60 min with increasing amounts

of the enzyme, either alone ( l ) or in the presence of 20 mM fructose l,&bisphosphate ( A ),

or 20 mM mannose 6-phosphate (0 ). The bound activity after a wash is plotted against the added

activity. The Scatchard's plot of the points is shorn at the right.

801


Table I. Binding of @-galactosidase of rat epididymal fluid to cellular membranes of the tissue

in the presence of carbohydrates, salts or detergents. Binding mixture contained 30 ug of mem-

branes proteins, 60 units of enzyme activity and the corresponding agent at the concentration

indicated in the table. The results are expressed as a percentage of the activity bound in the

absence of the agents (control). All solutions were adjusted to pH 6 with NaOH or HCl before

they were added to the binding mixture. Each value represents the mean of at least three assays.

The highest standard error obtained was 6%.

Added Bound Enzyme

Compound (X of control)

Added

Compound

Bound Enzyme

(X of control)

Sugar Phosphates (20 mM) Enzyme Substrates

fructose 6-phosphate 55

mannose &phosphate 78

glucose 6-phosphate 80

fructose l-phosphate 74

fructose 1,6-bisphosphate 27

fructose 2,6-bisphosphate 20

4-methylombelliferyl-

fl-galactoside (0.8 mM)

p-nitrophenyl-fl-

galactoside (10 mM)

Saccharides and Oerivates (100 mM)

fructose

mannose

glucose

galactose

lactose

sacarose

N-acetylglucosamine

CX-methyl-mannoside

manitol

Calcium and Chelating Agents

(2 Ml * CaCl

EDTA2

92

89

90

101

94

110

74

89

106

EGTA

Detergents (0.1%)

Triton X-100 89

Hyamine 2389 5

Salts (0.2 M)

KC1 34

NaCl 30

89

95

85

80

100

l Phosphate buffer was substituted by sodium acetate buffer 0.02 M, pH 6.

acid hydrolases by cultured fibroblasts (151, were more effective competitive inhibitors of

the @-galactosidase binding than mannose 6-phosphate (Fig. 2 and Table I). Moreover, fructose

l-phosphate which does inhibit the uptake mediated by the phosphomannosyl receptor (161, has

no effect in this binding (Table I). The coupling of the epididymal enzyme was resistant

to acidic pH (Fig. 3).

The binding of P-galactosidase was sensitive to ionic strength and to a cationic detergent

(Table I). Since the effect of salts and Hyamine was reversed by washing the membranes, it

seems that these agents dissociate the binding without destroying or solubilizing the affinity

sites of these structures.

The catalytic site of the enzyme does not seem to be involved in the binding, since the

substrates of the enzyme did not affect the coupling (Table I), and the competitive inhibitors

of the binding did not affect the enzymatic activity when they were incorporated in the reaction

mixture at 20 mM final concentration (data not shown).

802


I 1 8 I I -

r20mM t

50mM -0.25

/& Man &phosphate 7

VI 20mM Kiz63r6.5 In_ 0

Fru 1.6-bisphosphate

I U.“” 0.5

0.0

Fru 6-phosphate

Kiz 8.4t0.8

0 0.1 0 0.1 0.2

1 / FREE ACTIVITY ( units-' 1

Figure 2. Competitive inhibition of the binding of B-galactosidase to epididymal membranes by

fructose 1,6-bisphosphate, fructose 6-phosphate and mannose 6-phosphate. Bound activity was

measured in the presence of increasing concentrations of the Inhibitors, using from two to four

enzyme concentrations. Ki was estimated from each straight line as:

Ki = inhibitor concentration * slope / (slope - slope )

where slope The meson + standard 0

is the slope of the straight line in the absence of inhibitors. - error of the Ki are shown in the figure.

A very significant inhibition of the binding was obtained by periodic treatment of

@-galactosidase. The activity of the enzyme was sensitive to this treatment, however the

competition of inactive enzyme molecules for binding sites can not account for this inhibition

since the bound activity remained low when the data were corrected for the decrease caused

by the treatment (Fig. 4). The enzyme also lost its membrane affinity when it was exposed

to alkaline phosphatase (Fig. 4).

2 4 6 8

PH

Figure 3. Effect of pH on the binding of fl-galactosidase from rat epididymal fluid to membranes

of the tissue. The binding was performed using 30 ug of membrane proteins and 60 units of the

enzyme in 0.25 ml of 10 mM acetate buffer (0) or 10 mM phosphate buffer (0) adjusted to the

indicated pH with NaOH. After 60 min of incubation at 2O"C, the membranes were washed with 20

mM phosphate buffer pH 6. Bound activity at each ptl is expressed as a percentage of the highest

linked activity obtained in each assay. Vertical lines: standard error of three assays.

803

Vol. 143. No. 3. 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

( 0 30 60 0 30 60

UNITS ADDED

Figure 4. Effect of the treatment of epididymal @-galactosidase with alkaline phosphatase

(left) or metaperiodate (right) on its affinity for cellular membranes. Control ( l I and

treated enzymes ( 0 ) were incubated with 20 ug of membrane proteins, and the bound activity was

measured after washing the membranes. The presence of alkaline phosphatase in the binding assay

did not affect the binding of the untreated enzyme.

The recognizing sites of the membranes were extractable with 0.1% Triton X-100 and incorpor-

able into liposomes. They were destroied by trypsin treatment of the extracts but they were

not affected by proteolytic treatment of cellular membranes (Fig. 5).

DISCUSSION

The uptake of acid hydrolases by fibroblasts, and the binding of these enzymes to membranes

of the tissues lead to the identification of mannose Gphosphate as the common recognition

0 30 60 90 0 30 60 90

UNITS ADDED

Figure 5. Effect of trypsin treatment of cellular membranes (left) and of Triton X-100 extract

of cellular membranes (right) on their ability of binding @-galactosidase. Epididymal membranes

were incubated with ( 0 1 or without ( l ) 1 mg/ml of trypsin for 5 min at 37"C, and used in

binding assays. Treated membranes had roughly half the protein content of the untreated mem-

branes. The binding capacity of treated (1 mg/ml trypsin, 5 min, 37°C) ( 0 ), and untreated

( l 1 extracts was assayed after they were incorporated in liposomes. The binding capacity of

liposomes was also tested ( A 1. Eight micrograms of lipids containing 0, 5 and 2.8 ug of

proteins in the case of liposomes, liposomes + extract and liposomes + treated extract, respect-

ively, were used in binding assays.

804


marker in these proteins (191, and to the isolation from cellular membranes of the receptor

that recognizes this marker (21). Several models that assign an important role to these receo-

tors have been proposed (18,231. According to these models, the secreted enzymes are not

processed to their mature form and they conserve the phosphorylated mannosyl residue.

p-Galactosidase from rat epididymal fluid, which is secreted by the epithelium of the duct

(ll), binds indeed to membranes of the tissue with high affinity. This binding, like that

mediated by the phosphomannosyl receptor, is saturable, presents a Kd in the nM range, and

does not require calcium. However, the coupling is resistant to acidic pH, and is less inhibited

by mannose 6-phosphate than by fructose 6-phosphate derivates. This suggests that

fl-galactosidase is bound by a recognizing site different from the phosphomannosyl receptor.

The resistance to the unphosphorylated saccharides assayed and the absence of calcium requirement

indicate that the enzyme is not bound by other carbohydrate-recognizing proteins described

in animal tissues (17).

The nature of the membrane affinity of fl-galactosidase is not known, but it seems to depend

on the presence of a phosphorylated saccharide on the enzymatic molecule since it was inhibited

by phosphatase or periodic treatment of the enzyme. It is unlikely that fructose is involved

in the binding since it has not been found in the oligosaccharide moieties of glycoproteins.

However, many of the carbohydrate recognizing proteins can bind more than a saccharide; for

example the phosphomannosyl mediated endocytosis of various lysosomal enzymes can be inhibited

not only by mannose 6-phosphate but also by fructose l-phosphate (15). Then it seems possible

that a phosphosaccharide different from fructose 6-phosphate is recognized by the epididymal mem-

branes, and that fructose 6-phosphate derivates only resemble the conformation of the true

marker.

Trypsin treatment of the membranes does not significatively decrease their affinity for

P-galactosidase. This gives rise to some doubt about the protein nature of the binding sites.

However, the fact that a Triton X-100 extract of the membranes strongely increases the affinity

of liposomes for the enzyme, and that the trypsin treatment of the extract inhibites this in-

crease suggests that some intrinsic protein, with a trypsin resistant polypeptide facing the sur-

face of the membranes, may be involved in this binding.

The sensibility of the binding to high ionic strength indicates that electrostatic forces

are involved in the linkage of the enzyme. This raises the possibility that the interaction of

fl-galactosidase with the membranes is of non specific nature. However, the saturability, high

affinity and specific displacement by fructose 6-phosphate derivates of the binding suggest the

existance of a fi-galactosidase recognizing site. Moreover, the affinity strongely decreases

when the phosphosaccharide moieties of the enzyme are chemically or enzymatically attacked, while

805


the incubation with neuraminidase, which also affects the ionic charges of the enzyme molecules,

does not modify the binding properties of fl-galactosidase (data not shown).

The packaging of lysosomal enzymes mediated by phosphomannosyl receptors is thought to be

a general mechanism (IO). The presence of this receptor has been demonstrated by immunocytochem-

ical methods in various organs, including the epididymis (1). However, differences in the mech-

anism of translocation among tissues and among acid hydrolases of the same cell have been pro-

posed since patients with I-cell desease, which lack the enzymes required for the phosphorylation

of mannosyl residues, present normal levels of lysosomal enzymes in some organs (ZO), and a cel-

lular line that lacks phosphomannosyl receptors have normal, or almost normal levels of acid

hydrolases (22). A different mannose 6-phosphate-recognizing site, which requires calcium, has

been described in these cells (22).

The epididymis is an hormone-dependent organ, and its specific functions, as the maturation

and storage of spermatozoa are under androgen control (7). In the rat, the epithelial cells of

the duct synthesize high amounts of some lysosomal enzymes (41, and part of their activity is

secreted into the lumen (11). The activity of these enzymes is hormone-dependent in both, the

epithelium and the fluid. Then, the lysosomal apparatus of the epithelial cells seems to have

a particular function that includes the secretion of the enzymes into the lumen, and we wonder

if a special transport mechanism, involving a recognition marker different from mannose 6-phos-

phate, exists in the rat epididymis.

ACKNOWLEDGMENTS : We thank M.G. de Veca for her valuable technical assistance, Prof. E.C. de

Dubanced for correcting the manuscript, and Dr. H.H. Freeze for sending enzyme from Dictyostelium

discoideum, which was useful for interpreting our results. This work was supported by a grant

of the Consejo National de Investigaciones Cientificas y Tecnicas de la Rep3blica Argentina.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

REFERENCES

Brown, W.J., and Farquhar, M.G. (1984) Cell. 36, 295-307.

Von Figura, K., and Hasilik, A. (1986) Ann. Rev. Biochem. 55, 167-193,

Fischer, H.D., Gonzalez-Noriega, A., Sly, W.S., and Morre, D.J. (1980) J. Biol. Chem. 255,

9608-9615.

Chapman, D.A., and Killian, G.J. (1984) Biol. Reprod. 31, 627-636.

Gabel, C.A., Goldberg, D.E., and Kornfeld, S. (1982) J. Cell Biol. 95, 536-542.

Hasilik, A., and Neufeld, E.F. (1980) J. Biol. Chem. 255, 4937-4945.

Orgebin-Crist, M.C., Danzo, B.J., and Davies, J. (1975) Handbook of Physiology, sec. 7, vol.

V, pp. 319-338, Am. Physiol. Sot., Washington D.C.

Paigen, K., and Peterson, J. (1978) J. Clin. Invest. 61, 751-762.

LaRusso, N.F. (1979) Am. J. Dis. 24, 177-179.

Sly, W.S., Fischer, H.D., Gonzalez-Noriega, A., Grubb, J.H., and Natowicz, M. (1981) Methods

Cell Biol. 23, 191-214.

Mayorga, L.S., and Bertini, F. (1985) J. Androl. 6, 243-245.

806


12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

Barrett, A.J., and Heath, M.F. (1977) Lysosomes: a Laboratory Handbook, pp. 18-145,

Elsevier/North-Holland Biomedical Press, Amsterdam.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-

275.

Norden, A.G.W., Tennant, L.L., and O'Brien, J.S. (1974) J. Biol. Chem. 249, 7969-7976.

Ullrich, K., Mersmann, G., Weber, E., and Von Figura, K (1978) Biochem. J. 170, 643-650.

Shepherd, V.L., Freeze, H.H., Miller, A.L., and Stahl, P.O. (1984) J. Biol. Chem. 259,

2257-2261.

Ashwell, G., and Harford, J. (1982) Ann. Rev. Biochem. 51, 531-554.

Neufeld, E.F., Lim, T.W., and Shapiro, L.J. (1975) Ann. Rev. Biochem. 44, 357-376.

Kaplan, A., Achord, D.T., and Sly, W.S. (1977) Proc. Nat]. Acad. Sci. USA. 74, 2026-2030.

Owada, M., and Neufeld, E.F. (1982) Biochem. Biophys. Res. Commun. 105, 814-820.

Sahagian, G., Distler, J., and Jourdian, G.W. (1981) Proc. Natl. Acad. Sci. USA. 78,

4289-4293.

Hoflack, B., and Kornfeld, S. (1985) Proc. Natl. Acad. Sci. USA. 82, 4428-4432.

Sly, W.S., and Stahl, P. (1978) Transport of Macromolecules in Cellular Systems, pp.

229-244, Dahlem Konferenzen, Berlin.

807

β-galactosidase from rat epididymal fluid is bound by a recognition site attached to membranes of...

Documents