o-glycans chapter 8 april 6, 2004 jeff esko [email protected]
TRANSCRIPT
![Page 2: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/2.jpg)
Overview
• Types of O-linked glycans• T and Tn antigens• Glycosyltransferases involved in O-glycan
assembly• Mucins• Function of leukocyte membrane mucin• Tumor mucins
![Page 3: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/3.jpg)
O-Glycosidic Linkage
O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry)
-elimination
O
OH
H
H
HO
H
O
NAcHH
OH
NH2CHC
H2C
O Ser
GalNAc
![Page 4: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/4.jpg)
Examples of O-Glycans?
Ser/Thr
GalNAc
Mucins
Ser/Thr
Man
Yeast mannoproteins-dystroglycan
Ser/Thr
Fuc
NotchCoagulation FactorsFibrinolytic Factors
Ser/Thr
GlcNAc
Nuclear ProteinsCytoplasmic Proteins
![Page 5: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/5.jpg)
More O-glycans
Ser
Glc
Thr
Proteoglycans(Glycosaminglycans)
Ser
Xyl
Rho proteinsWorm CollagensDictyostelium proteins
Man Glc Gal GlcNAc
![Page 6: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/6.jpg)
Even more….
HydroxyLys
Gal
CollagenC1q complement
Plant glycoproteinsDictyostelium
HydroxyPro
Ara Gal GlcNAc
Tyr
Glycogenin
Glc
![Page 7: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/7.jpg)
Mucin-Type O-GalNAc Glycans
• Major vertebrate O-glycan
• Begins in cis-Golgi by attachment of GalNAc in -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
4
3
4
3
4
3
3
3
Ser/Thr
3 6
![Page 8: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/8.jpg)
Polypeptide GalNAc Transferases
• 12 members of mammalian ppGalNAcT family• Estimated size of family = 24• 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).
![Page 9: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/9.jpg)
Core 2GlcNAcT
Core 1GalT
Ser/Thr
3 6
Ser/Thr
3
Ser/Thr
Core 1 and Core 2 Synthesis
![Page 10: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/10.jpg)
Core 4GlcNAcT
Core 3GlcNAcT
Ser/Thr
3 6
Ser/Thr
3
Ser/Thr
Core 3 and Core 4 Synthesis
![Page 11: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/11.jpg)
Core 3
Ser/Thr
3
Core 4
Ser/Thr
3 6
Ser/Thr
3
Core 1
Ser/Thr
3 6
Core 2
Core 7
Ser/Thr
6
Core 6?
Ser/Thr
6
Core 5
Ser/Thr
3
Core 8
Ser/Thr
3
Unusual Core O-Glycan Structures
![Page 12: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/12.jpg)
T-antigens
• Tumor associated antigens• First one designated Thomsen-Friedenreich (TF)
antigen, later renamed T-antigen• Precursor (GalNAc-O-Ser/Thr) is called Tn-
antigen
Ser/Thr
Tn-antigen
Ser/Thr
3
T-antigen
3GalT
![Page 13: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/13.jpg)
Ser/Thr Ser/Thr
3
T-antigen
3GalT 6GlcNAcT 3 6
Ser/Thr
3
3
3 6
sialyl Tn-
antigen6
36
Tn-antigen
disialylT-antigen
Core 2
sialylT-antigen
ST3Gal-I, II, IV
ST6GalNAcIII, IV, I, II
ST6GalNAc-I ST6GalNAc-II, I
![Page 14: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/14.jpg)
Cosmc
• Tn-antigens accumulate due to loss of 3Gal transferase activity
• No mutations in 3GalT; message is expressed• Missing Cosmc (Core 1 3GalT -specific molecular chaperone,
Xq23• 25% identity, >40% homology to 3GalT• Absence of Cosmc results in proteosome degradation of
3GalT
Cosmc
Ju & Cummings (2002) PNAS 99:16613-8
Ser/Thr
Tn-antigen
Ser/Thr
3
T-antigen
3GalT
![Page 15: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/15.jpg)
Core 26GlcNAcT
Ser/Thr
3 6
Ser/Thr
3
Ser/Thr
Core 2 GlcNAc Transferases
• Three genes known, Core 2 6GlcNAcT I, II, III
• Two isoforms, 6GlcNAcT I and III, in lymphocytes and other non-epithelial cells
• One isoform, 6GlcNAcT II, specific for mucin secreting epithelia
![Page 16: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/16.jpg)
Outer Chain Assembly
• Sequential action of 4GalT and 3GlcNAcT gives rise to polylactosamine chains (Type II repeats)
• Type I repeats (Gal3GlcNAc4) also occur
4
3
4
3
4
3
3
3
Ser/Thr
3 6
• GlcNAc6Gal branches (I-antigen) can occur
• The ends of the chains are capped in -linked sugars, e.g. 3/4Fuc and 3/6sialic acids
• Terminal structures make up important blood group determinants, e.g. the Lewis antigens
![Page 17: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/17.jpg)
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 tend clustered - bottle brush
• Expressed by epithelial cells that line the gastrointestinal, respiratory, and genito-urinary tracts
![Page 18: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/18.jpg)
Mucins
• 11p15 family (MUC2, MUC5AC, MUC5B, MUC6) probably responsible for the formation of mucus layers
• 7q22 family (MUC3A, MUC3B, MUC12), 1q21 (MUC1), and 3q (MUC4, MUC13) are membrane mucins
Dekker et al. (2002) TIBS 27:126
![Page 19: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/19.jpg)
Lung Epithelium
Goblet cells in intestinal crypts
Mucin Production
![Page 20: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/20.jpg)
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
![Page 21: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/21.jpg)
Leukocyte Trafficking
Vascular Injury
HeparanSulfate
Endothelial Cells
P-selectin
SelectinLigands
Chemokines
Rolling Chemokine Activation of Integrins Extravasation
Cytokines
Integrin-ICAML-selectin
Infiltration of leukocytes into sites of inflammation depends on multiple carbohydrate-protein interactions
![Page 22: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/22.jpg)
Leukocyte Rolling
QuickTime™ and aSorenson Video decompressorare needed to see this picture.
Tim Springer, Harvard
![Page 23: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/23.jpg)
Cell Surface Mucin: PSGL-1
www.imech.ac.cn/mianlong/ ppt/ppt3.htm
![Page 24: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/24.jpg)
Lappänen et al. (2000) JBC 275:39569
![Page 25: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/25.jpg)
Leukocyte Trafficking Defects in Mice Lacking Core 2 GlcNAc Transferase
cells
/ml
Neutrophils Lymphocytes EosinophilsMonocytes
wt/∆wildtype ∆/∆
Leukocytosis (Ellies et al. (1998) Immunity 9:881-90)
![Page 26: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/26.jpg)
L-selectin chimeras were used to probe lymph node sections
Wildtype Null
Lymph Node High Endothelial Venules (HEVs)
Ellies et al. (1998) Immunity 9:881-90
![Page 27: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/27.jpg)
CarcinomaCell
Tumor cells express mucins that define ligands for selectin adhesion receptors
Platelets: P-selectinLeukocytes: L-selectinEndothelia: E- and P-selectins
Leukocyte
L
P
Activated
RestingPlatelet
Platelet
ACTIVATED ENDOTHELIUM
Neoplastic emboli can lodge in the small vessels
P E
MembraneBound Mucin
P
P
P
Tumor markers: CA19-9 (sLeA), CA125 (MUC16) and others
![Page 28: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/28.jpg)
Induced O-glycan Deficiencies in the Mouseand Biological Effects
Enzyme Phenotype
Polypeptide GalNAcT-1 B-lymphocyte deficiency in nodes
Polypeptide GalNAcT-8 None so far
Core 2 GlcNAc T-I Leukocytosis and defect in inflammation
![Page 29: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/29.jpg)
Questions
• What is the function of multiple polypeptide GalNAc transferases?
• Do various transferases within a family act on the same or different substrates?
• How is tissue specific expression of transferases regulated?
• How does competition of transferases for substrates determine the glycoforms expressed by cells and tissues?
• Would small molecule inhibitors of O-glycan formation prove therapeutically useful?
![Page 30: O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu](https://reader035.vdocuments.site/reader035/viewer/2022062318/5519accd550346c9608b4955/html5/thumbnails/30.jpg)
Core 1GalT
Ser/Thr
3
Ser/Thr
N-acetylgalactosaminides
O O
Core 1GalT