Glycosyltransferases in glycan biosynthesis

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Glycosyltransferases in glycan biosynthesis

• Major take home points
• Glycosyltransferases translate information
  contained in DNA to produce carbohydrate
  structures of defined sequence
• Carry out non-template driven synthesis of
  diverse families of glycans
• Majority of glycan classes synthesized
  simultaneously in organelles of the secretory
  pathway. (Exception -O-GlcNAc glycosylation of
  cytoplasmic and nuclear glycoproteins)
• Specificity of glycosyltransferases produced by a
  cell determine ultimate structures of glycans

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Alphabet 3 Carbohydrates
CARBOHYDRATES (Alphabet 3)
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Life Sugar-Coded Glycans are Positioned to
Mediate Flow of Information and Recognition Among
Cells
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Major classes of mammalian glycans

• Cell surface (secretory pathway)
• Glycoproteins
• N-linked to Asn
• O-linked to Thr/Ser
• GPI anchor
• Proteoglycans
• Glycolipids
• Intracellular (cytoplasmic/nuclear)
• O-linked GlcNAc

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Glycans synthesized in the secretory pathway
Figure 3.1
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Symbol Nomenclature
N-Linked
O-Linked
Modified IUPAC nomenclature
Key
Man
Gal
GlcNAc
Fuc
NeuAc
GalNAc
Glc
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General aspects of glycan structure
N-linked glycan
Key
Gal
GlcNAc
Fuc
NeuAc
Man
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Core and extension sequences on three classes of
glycans
Key Type 2 Extension sequence (N-acetyl-lact
osamine) Core sequence
Key
Gal
GlcNAc
Man
GalNAc
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Principles governing the synthesis of
glycoprotein glycans

• Glycans are are post-translational modifications
  of the secretory pathway, occuring in the
  endoplasmic reticulum (N-linked) and Golgi
  apparatus (N-linked and O-linked).
• Glycan structure is largely determined by the
  specificity of the glycosyltransferases expressed
  by a cell.
• Glycan structure at a given glycosylation site
  can be impacted by the adjacent protein
  structure.
• Each glycosylation site typically has a spectrum
  of related 'glycoforms'.

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Peptide sequence requirements for protein
glycosylation

• N-linked oligosaccharides
• Glycosylation motif Asn-X-Thr/Ser
• Glycosylation is initiated in the ER before
  protein folding.

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Subcellular organization of glycoprotein glycan
synthesis

• Synthesis of lipid-linked intermediate
• Initiation of N-linked glycosylation by transfer
  to nascent polypeptide
• Protein folding

Rough ER
Secretory Vesicle
Terminal glycosylation of N-linked and O-linked
glycans
Cis Golgi

• Processing of N-linked glycans
• Initiation of O-linked glycosylation

Trans Golgi
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Relationship between lipid linked intermediate
and N-linked glycans
Core
Lipid-linked precursor
Complex Type N-linked
Key
Man
Gal
GlcNAc
Fuc
NeuAc
GalNAc
Glc
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Synthesis of the N-glycan lipid-linked
intermediate
cytosol
SEC59
RFT1
ER lumen
OT
Glucose
Mannose
N-Acetylglucosamine
Cipollo, C.F. et al., Glycobiology, 2002, 12, 749


Dolichol pyrophosphate
Chantret et al., J. Biol. Chem. 2005, 280(10),
9236

Burda, P. and Aebi, M. Biochim. Biophys. Acta
1999, 1426, 239-257 (Review)

Frank, C.G. and Aebi, M. Glycobiology Advance
Access manuscript 2005

Cipollo, C.F. et al., J. Biol. Chem. 2001
276(24), 21828
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Processing of N-linked Glycans
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Processing of N-linked Glycans
Key
GlcNAc
Man
GlcNAc T
Glc
Glucosidase
Mannosidase
Gal
NeuAc
ER lumen
Gogi lumen
GlcNAc T
NeuAc T
Gal T
Mannosidase
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Some terminal sequences found on glycoprotein
and glycolipid glycans
Key
Fuc
Gal
NeuAc
GlcNAc
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Structure variation in N-linked glycans
Complex-Type Core
Type 1 Extension
Type 2 Extension
Key
GlcNAc
Fuc
GlcA
GalNAc
Glc
Man
NeuAc
Gal
Core sequence
Extension sequence
Sulfate
S
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Peptide sequence requirements for protein
glycosylation

• N-linked oligosaccharides
• Glycosylation motif Asn-X-Thr/Ser
• Glycosylation is initiated in the ER before
  protein folding.
• O-linked oligosaccharides (GalNAcaThr/Ser)
• Linkage is to Thr or Ser residues, no other
  defined motif determines site of glycosylation
• Sequence surrounding Thr/Ser and glycosylation of
  adjacent Thr/Ser residues can influence the
  activity of the GalNAc transferases (20
  isoenzymes) (Hassan, 2000).
• Glycosylation is initiated in the Golgi apparatus
  following protein folding.

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Key
GalNAc
GlcNAc
ER lumen
Gal
Synthesis of O-linked Glycans Occurs in the Golgi
Apparatus
NeuAc
Gogi lumen
NeuAc T
20
Mucin Type O-Linked Composite Structures
Core 1
Core 2
Core 3
Core 4
GlcNAc
Fuc
GlcA
GalNAc
Glc
Man
NeuAc
Gal
Key
Core sequence
Extension sequence
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Generalized glycosyltransferase reaction
Nucleotide-Sugar HO-Acceptor
Glycosyltransferase
Nucleotide
Sugar-O-Acceptor (Product)
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Glycosyltransferases
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Glycosyltransferases are type II membrane
glycoproteins with a common topology
Figure 3.2
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Glycosyltransferase families have conserved
sequence motifs for binding substrates
Sialymotifs of sialyltransferases
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Example of glycosyltransfese specificity(A
sialyltransferase ST3Gal III)
(Terminal sequence)
(Extension sequence)
ST3Gal III
Galb1-4GlcNAc
NeuAca2-3Galb1-4GlcNAc
CMP-NeuAc
CMP

• Sialyltransferase recognizes Galb1-4GlcNAc as an
  acceptor substrate, not Galb1-3GalNAc (like
  ST3Gal I).
• Sialyltransferase only makes a2-3 linkage not the
  a2-6 linkage (like ST6Gal I).

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Mutually exclusive glycosylation pathways
Figure 5.3
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Differential expression of sialyltransferases in
mouse tissues by glyco-gene microarray
Data compiled by Gene Microarray Core (E)
Consortium for Functional Glycomics http//www.f
unctionalglycomics.org
Key - Not detected Borderline level of
expression
Comelli (2006) Glycobiology 16,117
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Reversible O-linked glycosylation of cytoplasmic
and nuclear proteins
Reciprocal glycosylation and phosphorylation of
some proteins
Single sugar glycosylation
Hart, G. et al (2007) Nature 446, 1017-1022
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Some Questions-to start

• Subcellular localization of glycosylation. Why
  is N-linked glycosylation more likely than
  O-linked glycosylation to play an intrinsic role
  in protein folding (unrelated to quality control
  covered in the next lecture).
• Glycosyltransferase specificity I. Glycans carry
  information that mediate many biological
  processes. How does the specificity of
  glycosyltransferases play a role in translating
  the information carried by the genome to glycans
  synthesized by a cell.
• Glycosyltransferase specificity II. Glycans carry
  information that mediate many biological
  processes. How does expression of
  glycosyltransferase genes play a role in
  translating the information carried by the genome
  to glycans synthesized by a cell.

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Major N-linked glycans of a myeloma IgMHow can
glycan structures vary at different sites on the
same protein
IgM

NH2
COOH
364
462
171
563
332
variably present
Anderson et al. (1985)
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Major N-linked glycans of IgGWhy are the glycans
of IgG truncated?



N-glycans on IgG are truncated
Key
Fuc
Gal
GlcNAc
Man
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IgG Conserved N-linked oligosaccahride in the
Fc Domain

• How does the protein affect glycan structure?
• How does the N-linked glycan in the Fc domain
  influence Ab function?

Arnold et al. Annu. Rev. Immunol. (2007) 25, 2150
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Polysialic acid on NCAMWhy does only NCAM get
PSA?
Structures typically found on N-linked
oligosaccharides
Dead End
ST6Gal I
ST3Gal III/IV
ST8Gal II ST8Gal IV
Protein specific
Polysialic acid on NCAM (n2-200)
Angata et al. (1998) J.Biol. Chem. 273,
28524 Angata et al. (2000) J. Biol. Chem. 275,
22127
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Peptide determinants for polysialic acid
transferase
N-linked glycan PSA
NCAM
N-linked glycan - PSA
FN2
FN1
Acidic Patch E26 E24 D23 Required
for polysialylation
Fibronectin Domains
Mendriata et al. (2005) J. Biol. Chem. 280,32340
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Its on this side
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Fig. 5.7