4. If a GT uses a one-step mechanism it is retaining or inverting? 5. Name: Gal GlcNAc Neu5Gc Xyl GlcA

Transcription

1 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

2 O-Glycosylation GalNAc Yeast mannoproteins α-dystroglycan cadherins Man* Fuc* Notch Thrombospondin Factor IX Glc* GlcNAc* Mucins Notch Coagulation Factors Fibrinolytic Factors Nuclear Proteins Cytoplasmic Proteins * SEPARATE LECTURES ALSO: Proteoglycans, Hydroxyproline/Hydroxylysine Glycosylation After Esko, J

3 O-Glycosidic Linkage H HO OH OH H H H GalNAc O NAc O GalNAc α H O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry) β-elimination H 2 C C O CH Ser NH 2 After Esko, J

4 Glycan synthesis in a cellular context Most O-Glycosylated proteins are synthesized in the secretory pathway

5 O-Glycosylation GalNAc Man Fuc Glc GlcNAc

6 Mucin-Type O-GalNAc Glycans α3 α3 β4 β3 β4 β3 α3 β4 β3 β6 Major extracellular vertebrate O-glycan Begins in cis-golgi (ERGIC) by attachment of GalNAc in α- linkage to specific 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 After Esko, J

7 Polypeptide GalNAc Transferases 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). >20 ppgalnact family members Share structural features in active site Some have lectin (ricin) domain After Esko, J

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10 Core 1 and Core 2 Synthesis Tn Antigen Core 1 GalT (cis) T (TF) Antigen β3 Core 2 GlcNAcT β3 β6 ST6GalNAc1 (trans) Sialyl Tn Antigen α6 β3 α3 β3 α6 Disialyl T Antigen After Esko, J

11 From: Tongzhong Ju and Richard D. Cummings

12 Core 3 and Core 4 Synthesis Core 3 GlcNAcT Core 4 GlcNAcT β3 β3 β6 After Esko, J

13 Unusual Core O-Glycan Structures Core 1 Core 2 Core 3 Core 4 β3 β3 β6 β3 β3 β6 Core 5 Core 6? Core 7 Core 8 α3 β6 α6 α3 After Esko, J

14 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

15 Mucin Production Goblet cells in intestinal crypts Lung Epithelium After Esko, J

16 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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18 Antigenic epitopes found in mucins α3 α3 β4 β3 β4 β3 α3 β4 β3 β6

19 Type-3 H, A, and B antigens that form the O (H), A, and B blood group determinants on N- and O-glycans Essentials of Glycobiology Second Edition Chapter 13, Figure 6

20 A1 and A2 blood group antigens Essentials of Glycobiology Second Edition Chapter 13, Figure 9

21 Type-1 and -2 Lewis determinants Essentials of Glycobiology Second Edition Chapter 13, Figure 10

22 Lewis blood groups Essentials of Glycobiology Second Edition Chapter 13, Figure 11

23 Terminal GlcNAc residues are usually galactosylated Essentials of Glycobiology Second Edition Chapter 13, Figure 2

24 Poly-N-acetyllactosamine chains Essentials of Glycobiology Second Edition Chapter 13, Figure 3

25 i and I antigen synthesis Essentials of Glycobiology Second Edition Chapter 13, Figure 4

26 Synthesis and structure of the HNK-1 epitope Essentials of Glycobiology Second Edition Chapter 13, Figure 21

27 Structure and synthesis of the Galα1-3Gal antigen NOT IN HUMANS!!! Essentials of Glycobiology Second Edition Chapter 13, Figure 13

28 Synthesis of glycoproteins bearing terminal α2-3 sialic acid transferred by the ST3Gal family of sialyltransferases Essentials of Glycobiology Second Edition Chapter 13, Figure 17

29 Synthesis of the human Sd a or mouse CT antigen Essentials of Glycobiology Second Edition Chapter 13, Figure 16

30 Synthesis of α2-6 sialic acid on O-glycans and glycolipids by the ST6GalNAc family of sialyltransferases Essentials of Glycobiology Second Edition Chapter 13, Figure 18

31 Complex O-GalNAc glycans with different core structures Essentials of Glycobiology Second Edition Chapter 9, Figure 2

32 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

33 O-Glycosylation GalNAc Man Fuc Glc GlcNAc

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37 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)

38 O-Glycosylation GalNAc Man Fuc Glc GlcNAc

39 O-Glc Pathway Shao, L. et al. Glycobiology : ; doi: /glycob/cwf085

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41 Rumi is OGluT KDEL Retention Signal Temp. Sensitive Mutation

42 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)

43 O-Glycosylation GalNAc Man Fuc Glc GlcNAc

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46 POMT1 in the ER In Complex With POMT2 Uses Dol-Man As Donor

47 Alpha-Dystroglycan

48 Muscular Dystrophies associated with glycosylation of α DG (oversimplified) Disease Walker - Warburg Syndrome Muscle - Eye - Brain D isease Fukuyama - type MDC Limb - Girdle and MDC 1C Myodystrophy, myd MDC 1D Species Human Human Human Human Mouse Human Affected Gene POMT1 POMGnT1 Fukutin Fukutin - Related Protein LARGE Biochemical Lesion O - Man addition to Addition of GlcNac β 2 to O - Man Glycosyltransferase - like protein Glycosyltransferase - l ike Golgi protein Glycosyltransferase - like Golgi protein Biochemical Phenotype Decreased protein O - mannosylation Underglycosylated α - DG, uncapped O - Man Underglycosylated α - DG Underglycosylated α - DG Underglycosylated α - DG Other Disease causing genes: B4GAT1, POMGnT2, DPM1-3, ISPD, TMEM5.

49 Mammalian O-Mannosylation POMGnT1 POMGnT1+GnTVb POMGnT2

50 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

51 Other O-Mannosylated Proteins: 1. The Cadherin Family of Proteins -also involved in adhesion 2. RPTPβ -Receptor protein tyrosine receptor 3. OTHERS???

52 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)

53 O-Glycosylation GalNAc Man Fuc Glc GlcNAc

54 A. -6- P -6- P -6- P -6- P -1- P UDP - Glycogen PPP Glycolysis Hexosamine Biosynthetic Pathway (HBP) B. Complex Glycosylation O- - UDP - UDP - CMP - Key: = Glc = Fru = GlcN = GlcNAc = GalNAc = Neu5Ac

55 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

56 Classes of Proteins Modified By O-GlcNAc >1,000 Proteins Identified to Date Cytoskeletal Components Tau, Vinculin Transcription Factors c-myc, Sp1, Pax6 Hormone Receptors ERα&β Kinases & Other Signaling Molecules CKII, enos, IRS-2 Nuclear Pore Proteins NUP62, NUP155 Oncogenes & Tumor Suppresors p53, Rb Metabolic Enzymes GAPDH, Pyruvate Kinase Transcriptional and Translational Machinery RNA Pol II, EIF4A Viral Proteins SV40 T Antigen, v-erb-a Heat-Shock Proteins HSP90, α-crystallin

57 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)

58 A few more O-glycans GalNAc Man Fuc Glc Gal Xyl GlcNAc Hyl Ser Hyp β O-Xyl.precursor for GAGs Separate Lecture (Bergmann) O-GlcNAc (nucleocytoplasmic.separate lecture note: recent report of O-GlcNAc on Notch extracellular domain (different enzyme (eogt) than the intracellular one (OGT))

59 O-Glycosylation of Hyl

60 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?

61 After Esko, J The Glycosaminoglycans

62 O-Glycosylation -less studied (until recently?) -tools to study are underdevelopment -in many cases, clear functional effect