primer the 4-epimer of glucose once it is 2-n-acetylated is …glycobiology/lwoglycans2017.pdf ·...
TRANSCRIPT
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Primer
1. The 4-epimer of Glucose once it is 2-N-acetylated is called?2. If the SNFG shape of a monosaccharide is a diamond it is an ___?3. Which of the 11 common mammalian monosaccharides is:
a) a pentose?b) a deoxyhexose?c) never incorporated as a sugar nucleotide?
4. If a GT uses a one-step mechanism it is retaining or inverting?5. Name:
6. Shape/Color:
Gal GlcNAc Neu5Gc Xyl GlcA
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O-Glycosylation
Ser/Thr
GalNAc
Mucins
Ser/Thr
Man*
Yeast mannoproteinsα-dystroglycan
cadherins
Ser/Thr
Fuc*
NotchCoagulation FactorsFibrinolytic Factors
Ser/Thr
GlcNAc*
Nuclear ProteinsCytoplasmic Proteins
After Esko, J
Ser/Thr
Glc*
NotchThrombospondin
Factor IX
ALSO: Proteoglycans, Hydroxyproline/Hydroxylysine Glycosylation
* SEPARATE LECTURES
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O-Glycosidic Linkage
α
Ser
GalNAc
After Esko, J
O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry)
β-elimination
O
OH
H
H
HO
H
O
NAcHH
OH
NH2CHC
H2C
O
GalNAc
α
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Glycan synthesisin a cellular context
Most O-Glycosylatedproteins are synthesizedin the secretory pathway
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O-Glycosylation
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
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Mucin-Type O-GalNAc Glycans• Major extracellular vertebrate
O-glycan• Begins in cis-Golgi (ERGIC) by
attachment of GalNAc in α-linkage to specific Ser/Thr residues
• Assembly is simpler than N-linked chains - no lipid intermediate is used
• Always involves nucleotide sugars
• Always occurs by addition to non-reducing terminus or by branching
β4
β3
β4
β3
β4
α3
α3
α3
Ser/Thr
β3 β6
After Esko, J
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Polypeptide GalNAc Transferases
• >20 ppGalNAcT family members• Share structural features in active site• Some have lectin (ricin) domain
Regions in white, pink, red, and black represent, respectively, 0–29%, 30–69%, 70–99%, and 100% sequence identity (Hagen et al. (2003) Glycobiology 13:1R-16R).
After Esko, J
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Core 1 and Core 2 Synthesis
After Esko, J
T (TF)Antigen
TnAntigen
Core 2GlcNAcT
Core 1GalT(cis)
Ser/Thr
β3 β6
Ser/Thr
β3
Ser/Thr
α6
α3
ST6GalNAc1(trans)
Ser/Thr
SialylTn
AntigenSer/Thr
β3
Ser/Thr
β3 α6Disialyl
TAntigen
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From: Tongzhong Ju and Richard D. Cummings
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Core 4GlcNAcT
Core 3GlcNAcT
Ser/Thr
β3 β6
Ser/Thr
β3
Ser/Thr
Core 3 and Core 4 Synthesis
After Esko, J
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Core 3
Ser/Thr
β3
Core 4
Ser/Thr
β3 β6
Ser/Thr
β3
Core 1
Ser/Thr
β3 β6
Core 2
Core 7
Ser/Thr
α6
Core 6?
Ser/Thr
β6
Core 5
Ser/Thr
α3
Core 8
Ser/Thr
α3
Unusual Core O-Glycan Structures
After Esko, J
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Mucins are Heavily O-glycosylated
• Apomucin contain tandem repeats (8-169 amino acids) rich in proline, threonine, and serine (PTS domains)
• Glycosylation constitutes as much as 80% of mass and tends to be clustered - bottle brush
• Expressed by epithelial cells that line the gastrointestinal, respiratory, and genito-urinary tracts
After Esko, J
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Lung Epithelium
Goblet cells in intestinal crypts
Mucin Production
After Esko, J
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Mucins: Protective Barriers for Epithelial Cells
• Lubrication for epithelial surfaces• Modulate infection:
– Receptors for bacterial adhesins– Secreted mucins can act as decoys
• Barrier against freezing:– Antifreeze glycoproteins– [Ala-Ala-Thr]n≤40 with Core 1
disaccharides
After Esko, J
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Antigenic epitopes found in mucins
β4
β3
β4
β3
β4
α3
α3
α3
Ser/Thr
β3 β6
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Type-3 H, A, and B antigens that form the O (H), A, and B blood group determinants on N- and O-glycans
Chapter 13, Figure 6Essentials of Glycobiology
Second Edition
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A1 and A2 blood group antigens
Chapter 13, Figure 9Essentials of Glycobiology
Second Edition
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Type-1 and -2 Lewis determinants
Chapter 13, Figure 10Essentials of Glycobiology
Second Edition
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Lewis blood groups
Chapter 13, Figure 11Essentials of Glycobiology
Second Edition
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Terminal GlcNAc residues are usually galactosylated
Chapter 13, Figure 2Essentials of Glycobiology
Second Edition
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Poly-N-acetyllactosamine chains
Chapter 13, Figure 3Essentials of Glycobiology
Second Edition
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i and I antigen synthesis
Chapter 13, Figure 4Essentials of Glycobiology
Second Edition
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Synthesis and structure of the HNK-1 epitope
Chapter 13, Figure 21Essentials of Glycobiology
Second Edition
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Structure and synthesis of the Galα1-3Gal antigen
Chapter 13, Figure 13Essentials of Glycobiology
Second Edition
NOT IN HUMANS!!!
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Synthesis of glycoproteins bearing terminal α2-3 sialic acid transferred by the ST3Gal family of sialyltransferases
Chapter 13, Figure 17Essentials of Glycobiology
Second Edition
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Synthesis of the human Sda or mouse CT antigen
Chapter 13, Figure 16Essentials of Glycobiology
Second Edition
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Synthesis of α2-6 sialic acid on O-glycans and glycolipids by the ST6GalNAc family of sialyltransferases
Chapter 13, Figure 18Essentials of Glycobiology
Second Edition
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Complex O-GalNAc glycans with different core structures
Chapter 9, Figure 2Essentials of Glycobiology
Second Edition
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Questions
• What is the function of multiple polypeptide GalNAc transferases?
• How is tissue specific expression of transferases regulated?
• How does competition of transferases for substrates determine the glycoforms expressed by cells and tissues?
• What roles do chaperones play?
After Esko, J
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O-Glycosylation
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
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O-Fuc
• One of the clearest examples of glycosylation (Fringe) modulating signal transduction
• What other proteins carry O-Fuc and how does glycosylation modulate activity?
• How is glycosylation regulated?
• Separate Lecture (Haltiwanger)
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O-Glycosylation
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
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Shao, L. et al. Glycobiology 2002 12:763-770; doi:10.1093/glycob/cwf085
O-Glc Pathway
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Rumi is OGluT
KDEL Retention Signal
Temp. Sensitive Mutation
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O-Glc• Always a trisaccharide?• Glc & Xyl (except for proteoglycans) rarely
used on mammalian glycoproteins--why both here? Many of the same proteins as O-Fuc modifed, why?
• Role in Modulating Signaling? Regulated by enzymes or sugar nucleotide availability?
• Separate Lecture (Haltiwanger)
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O-Glycosylation
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
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POMT1in the ER
In Complex
WithPOMT2
Uses Dol-ManAs Donor
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Alpha-Dystroglycan
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Muscular Dystrophies associated with glycosylation of α – DG (oversimplified)
Disease Species Affected
Gene Biochemical
Lesion Biochemical Phenotype Walker - Warburg Syndrome Human POMT1 O - Man addition to
Ser/Thr Decreased protein O - mannosylation
Muscle - Eye - Brain D isease Human POMGnT1 Addition of GlcNac
β 2 to O - Man Underglycosylated α - DG, uncapped O - Man
Fukuyama - type MDC Human Fukutin Glycosyltransferase -
like protein Underglycosylated α - DG
Limb - Girdle and MDC 1C Human
Fukutin - Related Protein
Glycosyltransferase - l ike Golgi protein Underglycosylated α - DG
Myodystrophy, myd Mouse
MDC 1D Human LARGE Glycosyltransferase -
like Golgi protein Underglycosylated α - DG
Other Disease causing genes: B4GAT1, POMGnT2, DPM1-3, ISPD, TMEM5 ….
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Mammalian O-Mannosylation
POMGnT1 POMGnT1+GnTVb POMGnT2
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The ECM binding O-Man Structure (also the receptor for certain clades of arenaviruses including Lassa)
Praissman J L et al. eLife Sciences 2014;3:e03943
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Other O-Mannosylated Proteins:
1. The Cadherin Family of Proteins -also involved in adhesion
2. RPTPβ-Receptor protein tyrosine receptor
3. OTHERS???
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O-Man
• O-Man is clearly involved in CMD• What mammalian proteins (especially in the
brain) are O-Man modified besides α-DG?• What are the roles for M1, M2, and M3
glycans?• What is relationship between O-Man and O-
GalNAc?
• Separate Lecture (Wells)
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O-Glycosylation
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
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-6-P
-6-P
-6-P
-6-P
-1-P
UDP-
GlycogenPPP Glycolysis Hexosamine Biosynthetic Pathway
(HBP)
UDP-
- -O
UDP-
CMP-
Complex Glycosylation
A.
B.
Key:
= Glc
= Fru
= GlcN
= GlcNAc= GalNAc= Neu5Ac
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Features of O-GlcNAc• NOT elongated• Nucleocytoplasmic Proteins• Dynamic & Inducible• Enzymes for its Addition (OGT) and
Removal (O-GlcNAcase) are Nucleocytoplasmic
• OGT Knock-Out is Lethal at the ES cell stage– X-linked OGT, Females Heterozygotes are NOT mosaic
• Reciprocity with Phosphorylation (Yin-Yang) on Some Proteins
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Classes of Proteins Modified By O-GlcNAc ���>1,000 Proteins Identified to Date
❖ Cytoskeletal ComponentsTau, Vinculin
❖ Hormone ReceptorsERα&β
❖ Kinases & Other Signaling Molecules CKII, eNOS, IRS-2
❖ Nuclear Pore ProteinsNUP62, NUP155
❖ Oncogenes & Tumor Suppresors
p53, Rb
❖ Transcription Factorsc-myc, Sp1, Pax6
❖ Metabolic EnzymesGAPDH, Pyruvate Kinase
❖ Transcriptional and Translational Machinery
RNA Pol II, EIF4A
❖ Viral ProteinsSV40 T Antigen, v-Erb-a
❖ Heat-Shock ProteinsHSP90, α-crystallin
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O-GlcNAc Challenges• Assigning function to specific site on particular protein
(mapping sites)– Recent data has demonstrated this– Great example CAMKIV (O-GlcNAc in active site)– Another Example: Fox01 repression of gluconeogenic
enzymes in liver– Another Example: O-GlcNAc of Histone required for
mono-Ub modification• Regulation of OGT/OGA (PTM, protein:protein
associations)
• Separate Lecture (Wells)
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Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
Hyl HypSer
Gal Xyl
β
A few more O-glycans……
O-Xyl….precursor for GAGs…Separate Lecture (Bergmann)
O-GlcNAc (nucleocytoplasmic….separate lecturenote: recent report of O-GlcNAc on Notch extracellular domain (different enzyme (eOGT) than the intracellular one (OGT))
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O-Glycosylation of Hyl
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O-Glycosylation of Hyl
• Found on Collagen and Adiponectin (which has a “collagen-like” domain)
• Glycosylation Essential for Basement Membrane Formation in Tissues
• Modulates Collagen Cross-linking?• Other proteins with modification?
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After Esko, J
The Glycosaminoglycans
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���O-Glycosylation���
-less studied (until recently?)-tools to study are underdevelopment-in many cases, clear functional effect