essentials of glycobiology lecture 10 april 13th. 2004 ajit varki the sialic acids
TRANSCRIPT
Essentials of Glycobiology
Lecture 10
April 13th. 2004
Ajit Varki
The Sialic Acids
GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID
ANCHORANCHOR
Sialic Acids on
Vetebrate Glycans
OSer
OSer/Thr
NAsn
Ser-O-
OUTSIDE
INSIDE
NAsn
S S S
-O-SerS SSS S
EtnP
INOSITOL
P
NH
Ac
P
NS NS
Ac
S
2
P
GlycoproteinGlycoprotein
ProteoglycanProteoglycanN-LINKED CHAINSN-LINKED CHAINS
O-LINKED O-LINKED CHAINCHAIN
HYALURONANHYALURONAN
GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS HEPARAN SULFATEHEPARAN SULFATE
CHONDROITINCHONDROITIN SULFATESULFATE
Sialic AcidsSialic Acids
GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID
O-LINKED GlcNAcO-LINKED GlcNAc
The "Primary" Sialic Acids
H-C-OH
H-C-OH
H-C-OH
C OH-C-C-N
H
H
C C
C C
H
OH
H
OH
1
2
4 3
6
5
C-O
9
8
7
O
H O H
H
H
-C C
H-C-OH
H-C-OH
H-C-OH
C OHO
HH
H
C C
H
OH
H
OH
1
2
4 3
6
5
C-O
9
8
7
O
-
-D-KDN-D-Neu5Ac
Thought to be the metabolic precursors of all other sialic acids
N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-
D-glycero-D-galacto-nonulosonic acid)“Neu5Ac” “NANA”, “NeuAc”
KDN (2-keto-3-deoxy-D-glycero-
D-galacto-nonulosonic acid)
NUCLEUS
Pathways of N-acetylneuraminic Acid (Neu5Ac) metabolism
PLASMA MEMBRANESECRETORY PATHWAYS
LYOSOSOMES
ER
Newly synthesized glycoconjugate
GOLGI
Neu5AcManNAc
CYTOSOL
CELL MEMBRANE
CMP-Neu5Ac
1
1 CMP-Sialic Acid Synthase
5
5 Sialic Acid Exporter
4 Lysosomal Sialidase
Neu5Ac
4-Neu5Ac
ENDO/PINOCYTOSIS
-Neu5Ac
-Neu5Ac
UDP-GlcNAc
2 CMP-Sialic acid transporter3 Sialyltransferases
-Neu5Ac2
3
ManNAc
Neu5Ac
Feedback Inhibition
Sialyltransferases in the Mammalian Genome
Enzyme
Acceptor
Linkage
ST3Gal-IST3Gal-IIST3Gal-IIIST3Gal-IVST3Gal-VST3Gal-VI
ST6Gal-IST6GalNAc-IIST6GalNAc-III
ST6GalNAc-VST6GalNAc-IV
ST8Sia-IST8Sia-IIST8Sia-IIIST8Sia-IVST8Sia-V
Sialic acidGalactose N-AcetylgalactosamineGalactose
2-32-6 2-6 2-8
ST6GalNAc-VI
ST6GalNAc-I
ST8Sia-VI
ST6Gal-II
Jamey Marth
Biological Roles of Sialic Acids
Structural/Physical Roles
INTRINSIC RECEPTOR
SELF
IntrinsicRecognition
“Self”
SiglecsFactor HSelectins
Uterine AgglutininLaminins
SIALYLATED OLIGOSACCHARIDE =
M
EXTRINSIC RECEPTOR
M = Micro-organism or Toxin
ExtrinsicRecognition“Non-self”
InfluenzaMalariaCholera
HelicobacterMycoplasma
RotavirusPolyoma virusCoronavirusPertussis
Tetanus etc.
SELF
MolecularMimicry
E.ColiGonococcus
MeningococcusCampylobacterTrypanosoma
StreptococcusEtc.
DID THE SIALIC ACIDS APPEAR LATE IN EVOLUTION?
Adapted from : Schauer (1982)
Exceptions
• Certain bacteria (mostly animal pathogens)
• Certain protozoa (mostly animal pathogens)
• Certain fungi (animal pathogens)
• Rare cultured insect cell lines?
• Rare insect embryo stages?
• Octopus and Squid brain?
MammaliaAvesReptiliaAmphibiaDipnoi?TeleosteiChondrosteiElasmobranchiAgnathaCephalochordataUrochordataHemichordataEchinodermata
InsectaCrustaceaArachinidaAnnelidiaMolluscaBrachiopodaBryozoa
DETECTED
NOTDETECTED
De
ute
ros
tom
esP
roto
stom
es
Microbial genes in the human genome: lateral transfer or gene loss?
Salzberg et al. Science 2001, 292, 1903-1906
• 40 Candidate Genes fulfilling criteria suggesting Lateral transfer from Vertebrates to Bacteria
OUR ANALYSIS OF THE LIST:
• 7 of the 40 are involved in sialic acid biosynthesis, turnover or degradation
• Thus, pathway involving ~0.1% of the human genome represents almost 20% of the potential examples of lateral gene transfer between vertebrates and bacteria!
Phylogenetic relationships of enzymes
involved in the
metabolism of sialic acids
Neu5Ac/bacteria
GlcNAc-6-phosphate
ManNAc
Neu5Ac
CMP-Neu5Ac
ManNAc-6 -phosphate
Pi
PEP
Pi
CTP
PPi
HOMOLOGYHOMOLOGY
PhosphatasePhosphatase
GlcNAc-6-GlcNAc-6-phosphatephosphate
2-epimerase2-epimerase
Neu5AcNeu5Acsynthetasesynthetase
CMP-Neu5AcCMP-Neu5Acsynthetasesynthetase
Neu5Ac/Vertebrates
UDP-GlcNAc
ManNAc
Neu5Ac-9-phosphate
Neu5Ac
CMP-Neu5Ac
ATP
ADP
UDP
PEP
Pi
Pi
CTP
PPi
ManNAc-6 -phosphate
ManNAc kinaseManNAc kinase
UDP-GlcNAcUDP-GlcNAc2-epimerase2-epimerase
Neu5Ac-9-Neu5Ac-9-phosphatephosphatesynthetasesynthetase
CMP-Neu5AcCMP-Neu5Acsynthetasesynthetase
Neu5Ac-9-phosphateNeu5Ac-9-phosphatephosphatasephosphatase
Angata and Varki Chemical
Reviews 102, 439-469, 2002.
PlantsFungiEukaryaArchaeaCommon ancestor of cellular life
EuryarchaeotaCrenarchaeotaProtozoaProtostomesDeuterostomesSpirochetesChlamydiaThermus/DeinococcusCyanobacteriaAquifexGram-positiveHigh G+CGram-positiveLow G+C Gram-negativeBacteria"Universal tree" of cellular
organisms and occurrence of sialic acids
BiochemicalEvidence
GeneticEvidence
Phylogenetic relationships of enzymes
involved in the
metabolism of sialic acids
and KDO
Neu5Ac/bacteria
GlcNAc-6-phosphate
ManNAc
Neu5Ac
CMP-Neu5Ac
ManNAc-6 -phosphate
Pi
PEP
Pi
CTP
PPi
KDO/bacteria
Ara-5-phosphate
KDO-8-phosphate
KDO
CMP-KDO
PEP
Pi
Pi
CTP
PPi
PhosphatasePhosphataseHOMOLOGYHOMOLOGY
PhosphatasePhosphatase
GlcNAc-6-GlcNAc-6-phosphatephosphate
2-epimerase2-epimerase
Neu5AcNeu5Acsynthetasesynthetase
CMP-Neu5AcCMP-Neu5Acsynthetasesynthetase
Neu5Ac/Vertebrates
UDP-GlcNAc
ManNAc
Neu5Ac-9-phosphate
Neu5Ac
CMP-Neu5Ac
ATP
ADP
UDP
PEP
Pi
Pi
CTP
PPi
ManNAc-6 -phosphate
ManNAc kinaseManNAc kinase
UDP-GlcNAcUDP-GlcNAc2-epimerase2-epimerase
Neu5Ac-9-Neu5Ac-9-phosphatephosphatesynthetasesynthetase
CMP-Neu5AcCMP-Neu5Acsynthetasesynthetase
Neu5Ac-9-phosphateNeu5Ac-9-phosphatephosphatasephosphatase
CMP-KDOCMP-KDOsynthetasesynthetase
KDO-8-phosphateKDO-8-phosphatesynthetasesynthetase
Angata and Varki Chemical
Reviews 102, 439-469, 2002.
Possible Scenarios for the Phylogenetic Origins of Sialic Acids
Scenario 1: Ancient inventionBacteriaArchaeaEukaryaInvention of SiaDeuterostome Lineage
Loss or partial loss in many lineages
BiochemicalEvidence
GeneticEvidence
Lateral TransferAmongst
Prokaryotes
Possible Scenarios for the Phylogenetic Origins of Sialic Acids
Scenario 2: Invention in bacteriaInvention of SiaBacteriaArchaeaEukaryaDeuterostome Lineage
Loss or partial loss in many lineages
BiochemicalEvidence
GeneticEvidence
Lateral TransferAmongst
Prokaryotes
Lateral Transfer
toEarly
Animal Ancestor
Possible Scenarios for the Phylogenetic Origins of Sialic Acids
Scenario 3: Invention in early eukaryoteInvention of SiaBacteriaArchaeaEukaryaDeuterostome Lineage
Loss or partial loss in many lineages
BiochemicalEvidence
GeneticEvidence
Lateral TransferAmongst
Prokaryotes
Lateral Transfer
ToProkaryotes
fromEarly
Animal Ancestor
Biological Roles of Sialic Acids
Structural/Physical Roles
INTRINSIC RECEPTOR
SELF
IntrinsicRecognition
“Self”
SiglecsFactor HSelectins
Uterine AgglutininLaminins
SIALYLATED OLIGOSACCHARIDE =
M
EXTRINSIC RECEPTOR
M = Micro-organism or Toxin
ExtrinsicRecognition“Non-self”
InfluenzaMalariaCholera
HelicobacterMycoplasma
RotavirusPolyoma virusCoronavirusPertussis
Tetanus etc.
SELF
MolecularMimicry
E.ColiGonococcus
MeningococcusCampylobacterTrypanosoma
StreptococcusEtc.
Terminal Sialic acids, Oligosialic acids, Polysialic acidsand the Enzymes that can degrade them
SIALIDASE (NEURAMINIDASE)
Gangliosideor
N-Glycanor
O-glycanOr
BacterialGlycan
Sia2-3(6)Gal
Polysialic Acid
Oligosialic Acid
Terminal Sialic Acid
Sia2-8Sia2-3(6)Gal
Sia2-8Sia2-8Sia2-8Sia2-8Sia2-3(6)Gal
ENDOSIALIDASE (ENDO-NEURAMINIDASE)
The Neural Cell Adhesion
Molecule(N-CAM)
Expression of Polysialic Acid on N-CAM also
affects Cell-Cell Interactions involving
Other Adhesion Molecules
WHY DID THE NCAM-PSA MECHANISM EVOLVE?
MOLECULAR MECHANISM FOR GLOBAL REGULATION:
PSA evolved to allow regulation of an expanded array of different CAMs without the requirement that all of these CAMs be affected by the same signaling pathways
EXPANDED NEED FOR PLASTICITY:Migration of precursor cellsPathfinding by large groups of axonsRetention of plasticity in certain adult brain tissues
Evolutionary History of N-CAMNCAM/apCAM/FasII
Progenitor
MouseNCAM
ChimpanzeeNCAM
AplysiaapCAM
DrosophilaFasII
VERTEBRATES+PSA, +NCAM-2
INVERTEBRATES(no PSA)
More adult PSA(brain)
More “hardwired”More “plastic”
HumanNCAM
Fly: “most of my genes are like his”
Albert: “vive la différence”
SELF
Ligands forIntrinsic
Receptors
SiglecsFactor HSelectins
Uterine AgglutininLaminins
INTRINSIC RECEPTORINTRINSIC RECEPTOR
SIALYLATED GLYCAN = M = Micro-organism/Toxin
SELF
M
EXTRINSIC RECEPTOREXTRINSIC RECEPTOR
InfluenzaMalariaCholera
HelicobacterMycoplasma
RotavirusPolyoma virusCoronavirus
Pertussis Tetanus etc.
Ligands for ExtrinsicReceptors
Biological Roles of Sialic Acids
Structural/Physical Roles
MolecularMimicry
E.ColiGonococcus
MeningococcusCampylobacterTrypanosomaStreptococcus
Etc.
?
INFLUENZA A & B VIRUSES BIND TO CELL SURFACES VIAA HEMAGGLUTININ THAT RECOGNIZES SIALIC ACIDS
THE "RECEPTOR-DESTROYING ENZYME"
IS A NEURAMINIDASE (SIALIDASE)
LINKAGE TOUNDERLYINGSUGAR CHAIN
HEMAGGLUTININ
NEURAMINIDASE
INFLUENZAA OR B VIRUS
99
Sialic Acids Restrict Complement Activation on Cell Surfaces
FLUIDPHASE
Requires side chain
of Sia
C3 Bb C3b
P
Factor H
C3b iC3b
Bb
I
C3bBb C3b
C5
C5b
P
MembraneAttackComplex
ACTIVATING SURFACE
Sia
Factor H
C3b iC3b
I
Bb
NON- ACTIVATINGSURFACE
Sialic Acid-binding Lectins are Widespread in NatureGROUP SUB-GROUP EXAMPLEVertebrate C - type lectins L-Selectin
I - type lectins CD22 (Siglec-2)Unclassified Complement Factor H
Bacterial Adhesin E.coli S-adhesins Toxin Vibrio cholerae toxin
Viral Hemagglutinin Influenza A virusHemagglutinin-esterase Influenza C virusHemagglutinin-neuraminidase Sendai virus
Protozoal Plasmodium P. falciparum erythrocyte-binding antigen
Tritrichomonas Tritrichomonas mobilensis Lectin.Crustacean Crab Limulin
Lobster L-Agglutinin IPrawn Monodin
Arachnid Scorpion Vaejovis spinigerus lectinBeetle Allomyrina dichotoma lectinsSpider Aphonopelma lectin
Mollusc Slug Limax flavus agglutininSnail AchatininH
Plant Seed Maackia Amurensis lectinBark Sambucus Nigra lectinRoot Tricosanthes japonicum lectin
Fungal Mushroom Hericium erinaceum lectin
Natural substitutions can change the mass and shape of the Sialic Acid molecule
Carbon Oxygen Nitrogen Hydrogen
8-O-METHYL-7,9 DI-O-ACETYL-7,9 DI-O-ACETYL-N-GLYCOLYLN-GLYCOLYLNEURAMINIC
ACID
N -ACETYLNEURAMINIC ACID
8877
11
44
22
33
55
66
99
8877
11
44
22
33
55
66
99
THE SIALIC ACIDS
R2
R2
R5
R5N-acetyl
Hydrogen or:-linkage to: -Gal (-3 -4 -6) or -GalNAc (-6) or -GlcNAc(-4 -6) or -Sialic Acid (-8 -9) or-linkage to CMP orAbsent in: - 2,3dehydro or - 2,7anhydro (double-bond when R2 absent)
R5
R5
R5
N-glycolyl
Hydroxyl
Amino
*
-at physio-logical pH,ionized or
lactonized orlactamized
R11122
3344
55
6677
88
99
*
CARBON OXYGEN HYDROGEN
R1
= hydrogen or:
R8
R9
R7
R4
R8R9 R7 R4
Acetyl
R8R9 R7 R4
Phosphate R9
Methyl
R8
Sulfate R8
LactylR9
Nomenclature and Abbreviations
Combinations of:
Neu = neuraminic acid
KDN = 2-keto-3-deoxy-nonulosonic acid.
Ac = acetyl, Gc = glycolyl, Me = methyl, Lt = lactyl, S=sulfate
Examples:
N-glycolyl-neuraminic acid = Neu5Gc
9-O-acetyl-8-O-methyl-N-acetyl-neuraminic acid = Neu5,9Ac 8Me
7,8,9-tri-O-acetyl-N-glycolyl-neuraminic acid = Neu5Gc7,8,9Ac
Uncertain of the type of the sialic acid ? Use generic abbreviation Sia
Sialic acid of unknown type with O- acetyl at 9-position = Sia9Ac
3
2
O-ACETYL ESTERS AT THE 7- AND 9-POSITIONS
OF SIALIC ACIDS
7
9
8
7
9
8
7
9
8
7-O-ACETYL-N-ACETYL-NEURAMINIC ACID
O-acetyltransferases
9-O-acetylesterases
O-acetyl MigrationMigrase Enzyme?
INFLUENZA A VIRUSINFLUENZA A VIRUS
N-ACETYL-NEURAMINIC ACID
INFLUENZA C INFLUENZA C CORONAVIRUSESCORONAVIRUSES
9-O-ACETYL-N-ACETYL-NEURAMINIC ACID
INFLUENZA C AND CORONAVIRUS HEMAGGLUTININ-ESTERASES SPECIFICALLY RECOGNIZE
9-O-ACETYLATED SIALIC ACIDS
LINKAGE TOUNDERLYING
SUGAR
THE "RECEPTOR-DESTROYING ENZYME" IS A SIALIC ACID-SPECIFIC 9-O-ACETYL-ESTERASE
AT 37OC, THE ESTERASE ACTIVITY IS DOMINANT
INFLUENZAC VIRUS
HEMAGGLUTININ
ESTERASE
9
Ser
Sialic Acid-binding Proteins recognize specific features of Sialic Acids in natural ligands: some examples
Underlying Importance for Recognition Lectins Linkage Saccharide Carbo- N-Acyl Side 9-O-Acetylation
xylate Group Chain of Side chain
Selectins Sia2-3 Gal1-(3)4GlcNAc1- yes No No No? 1 Fuc(3)4
CD22 (Siglec-2) Sia2-6 Gal1-4GlcNAc1 yes yes yes Blocks
Sialoadhesin (Siglec-1) Sia2-3 Gal1-4GlcNAc1 yes yes yes Blocks
Complement Factor H Sia2-? ? yes no? yes Blocks
P. falciparum Sia2-3 Gal1-4GlcNAc1- ? no? ? Blocks? merozoite lectin
Influenza A Sia2-3 ? yes variable yes Blocks Hemagglutinin Sia2-6
Influenza C Hemagglutinin-esterase Sia2-X - yes no? yes Required
Carbon Oxygen Nitrogen Hydrogen
Two Major Kinds of Sialic Acidsin Mammalian Cells
N-ACETYLNEURAMINIC ACID
(Neu5Ac)88
77
1122
33
55
66
99
LINKAGE TOUNDERLYING
SUGAR CHAIN
44
N-GLYCOLYLNEURAMINIC ACID
(Neu5Gc)
8877
11
44
22
33
55
66
99
LINKAGE TOUNDERLYING
SUGAR CHAIN
“Great Apes”
Evolutionary Relationships
amongst Humans and the
Great Apes
10
5
Mil
lio
ns
of
Yea
rs B
efo
re P
rese
nt* 0
*Precise Timing Uncertain
Gorilla gorilla
GorillaPan paniscusBonobo
Pan troglodytes Chimpanzee
MEAN Amino AcidDifference
~0.5%<1.0%
Homo sapiens Human
Pongo pygmaeusOrangutan
Neu5Ac Neu5Gc
Genetic Mutation
Causing lossOf Neu5Gc
R3
R2
The Sialic AcidsCARBON
NITROGEN
OXYGEN
HYDROGEN
11
22
33
44
55
66
77
88
99
= H, ACETYL (4,7,8,9), LACTYL (9), METHYL (8), SULFATE (8,9), PHOSPHATE (9),
ANHYDRO (4,8 or 2,7), SIALIC ACID (8,9), FUCOSE(4), GLUCOSE(8), OR GALACTOSE(4)
= N-ACETYL, N-GLYCOLYL, N-GLYCOLYL-O-ACETYL, AMINO, HYDROXYL
= Gal (3/4/6), GalNAc(6), GlcNAc(4/6), Sia (8/9) or 5-O-Neu5Gc, (absent in 2,6 / 2,7 ANHYDRO)
R1
R1
R1
R1
R1
R2
R3