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Chemical Tools for Studying and Perturbing Glycans

Chemical Tools for Studying and Perturbing Glycans. Lecture 41 Carolyn R. Bertozzi UC Berkeley. Carbohydrate-. specific receptor. Unnatural. Cell surface. glycoconjugates. Metabolic. interconversions. Monosaccharide. "building blocks". Glycosyltransferase or glycosidase inhibitors.

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Chemical Tools for Studying and Perturbing Glycans

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  1. Chemical Tools for Studying and Perturbing Glycans Lecture 41 Carolyn R. Bertozzi UC Berkeley

  2. Carbohydrate- specific receptor Unnatural Cell surface glycoconjugates Metabolic interconversions Monosaccharide "building blocks" Glycosyltransferase or glycosidase inhibitors Glycoconjugate assembly Chemical approaches for perturbing cellular glycans Substrates ER/Golgi Cytosol Bertozzi, C. R.; Kiessling, L. L. Science2001, 291, 2357.

  3. Lecture Outline 1. Inhibitors of glycosyltransferases and glycosidases - Natural products - Rational design - Library screening 2. Chemical activation of glycosyltransferases 3. Substrate-based methods for perturbing glycosylation - Glycoside primers - Metabolic oligosaccharide engineering

  4. O H N O H H O O N O R C H N H O O C H 2 O O H H N H A c H H O H O H O O H H O O H Tunicamycin - An inhibitor of N-linked glycan biosynthesis • Blocks the transfer of GlcNAc-1-P from UDP-GlcNAc to dolichyl-P (GPT) • Resistant mutants overproduce GPT • Km for UDP-GlcNAc is ~3 x 10-6 M, whereas the Ki value for tunicamycin is ~5 x 10-8M

  5. 2 a ± 3 3 a a 3 3 a a 2 2 2 2 2 2 2 2 2 a a a a a a a a a 2 2 2 3 6 3 6 3 6 3 6 3 6 a a a a a a a a a 2 a a a a b 3 6 3 6 3 6 3 6 3 6 3 6 a a a a a a a a a a a a 4 4 4 4 4 4 b b b b b b G l c N A c T I 4 4 4 4 - g l u c o s i d a s e I - g l u c o s i d a s e I I - m a n n o s i d a s e I - m a n n o s i d a s e I I 4 4 a a a a b b b b b b A s n A s n A s n A s n A s n A s n Plant Alkaloids - Natural Inhibitors of Glycosidases Australine Swainsonine Kifunensin Deoxynojirimycin Castanospermine Deoxymannojirimycin

  6. H O HO O H Plant Alkaloids CH2OH O H HO HO O H H O H N H N OH O H N H O N O H H C H O H O H 2 Australine Castanospermine Deoxymannojirimycin Swainsonine a-Glucosidase I a-Glucosidase I and II a-Mannosidase I a-Mannosidase II • Alkaloids contain polyhydroxylated ring systems that mimic the orientation of hydroxyl groups in the natural substrates • Protonation of the ring nitrogen may mimic the positive charge developed on the ring oxygen during the hydrolytic reaction

  7. Glycosyltransferase inhibitors based on substrates/transition states

  8. Problems with most rationally designed inhibitors • Lack of activity in cells (poor bioavailability) • Lack of selectivity Pharmaceutical approach: High-throughput screen followed by medicinal chemistry optimization of hits

  9. Tools for studying N-linked glycosylation Conserved core Consensus sequence N-X-S/T Affinity reagents ConA, LPHA Small molecule inhibitors Tunicamycin Enzymes for cleavage PNGase F

  10. Consensus sequence None Nothing general Affinity reagents Small molecule inhibitors None Enzymes for cleavage Nothing general Challenges in studying O-linked glycosylation Conserved core

  11. The polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) initiate mucin-type O-linked glycosylation protein substrate ppGalNAcTs (~24 in human) Tabak et al. UDP-GalNAc elaborating glycosyltranferases Complex mucin-type O-linked glycoproteins

  12. diphosphate mimic sugar substitute nucleotide sugar substrate uridine uridine-based library > 400 commercial aldehydes uridine analogs Design and synthesis of a 1338-member uridine-based library Winans, K. A.; Bertozzi, C. R. Chem. Biol.2002, 9, 113-129

  13. KM of donor (mM) 22 25 32 69 51 20 10 20 25 20 IC50 1-68A (mM) 24 18 38 20 26 27 6 32 > 500 > 500 IC50 2-68A (mM) 21 15 40 30 20 22 7 39 > 500 > 500 mppGalNAcT-1 mppGalNAcT-2 mppGalNAcT-3 mppGalNAcT-4 rppGalNAcT-5 rppGalNAcT-7 rppGalNAcT-10 mppGalNAc-11 b1-4GalT-1 a1-3GalT Uridine analogs identified as broad spectrum ppGalNAcT inhibitors Ki of UDP = 380 mM 1-68A Ki = 7.84 + 0.96 mM (T-1) 2-68A Ki = 7.50 + 1.02 mM (T-1) Hang, H. C., et al. Chem. Biol.2004, 11, 337-345

  14. 1-68A inhibits O-linked but not N-linked glycosylation in Jurkat cells 160 140 MAA (a2,3NeuAc) 120 ConA (N-linked) 100 %MFI 80 VVA (O-linked) 60 HPA (O-linked) 40 20 0 0 50 100 150 200 250 [inhibitor] (mM)

  15. Lecture Outline 1. Inhibitors of glycosyltransferases and glycosidases - Natural products - Rational design - Library screening 2. Chemical activation of glycosyltransferases 3. Substrate-based methods for perturbing glycosylation - Glycoside primers - Metabolic oligosaccharide engineering

  16. Glycosyltransferases are residents of the Golgi compartment cis medial trans TGN

  17. Anatomy of a prototypical glycosyltransferase N-terminal cytosolic tail (C) Cytosol Transmembrane Domain (T) Golgi membrane Golgi lumen Stem region (S) C-terminal catalytic domain (CAT) CTS = Encodes Golgi localization CAT = Encodes catalytic activity

  18. Golgi localized Golgi localized Secreted Golgi localized Small molecule binding proteins Exploiting the requirement of Golgi localization for small molecule switching of enzyme activity Kohler, J. J.; Bertozzi, C. R. Chem. Biol.2003, 10, 1303-1311

  19. Rapamycin mediates association of FKBP and FRB Rapamycin J. Liang, J. Choi, & J. Clardy. Acta Cryst. (1999) D55, 745-752

  20. { FucT7 Fucosyltransferase 7 is involved in selectinligand biosynthesis Leukocyte L-Selectin Endothelial cell

  21. N C Tail TM Stem FRB or FKBP n = 1-3 Rap N C FKBP or FRB Catalytic domain n = 1-3 FucT7 constructs for CHO cell transfection N C Tail TM Stem Catalytic domain CAT (39-342) CTS (1-51)

  22. FucT7 activity in CHO cells can be monitored by the expression of sialyl Lewis x Sialyl Lewis x (detected with mAb HECA-452)

  23. No Rapamycin: FucT7 is “off” sLex

  24. + Rapamycin: FucT7 is “on” sLex

  25. Lecture Outline 1. Inhibitors of glycosyltransferases and glycosidases - Natural products - Rational design - Library screening 2. Chemical activation of glycosyltransferases 3. Substrate-based methods for perturbing glycosylation - Glycoside primers - Metabolic oligosaccharide engineering

  26. Carbohydrate- specific receptor Unnatural Cell surface glycoconjugates Metabolic interconversions Monosaccharide "building blocks" Glycoconjugate assembly Chemical approaches for perturbing cellular glycans Substrates ER/Golgi Cytosol Bertozzi, C. R.; Kiessling, L. L. Science2001, 291, 2357.

  27. • Prepare compounds that resemble biosynthetic intermediates • Conjugate to a hydrophobic aglycone to enhance uptake and activity • Alkylation or acylation also improve bioavailability H H O H O O H O H O H H H Glycoside Primers - Substrate Mimicry b-Napthyl xyloside Sarkar et al. (1995) Proc. Natl. Acad. Sci. USA 92: 3323

  28. Proteoglycan 6 S 6 S O O 2 S N S N S 6 S 6 S Hydrophobic O Xylose N S N S 2 S Aglycone 6S 6S O NS NS 2S 6S 6S O Xyloside NS NS 2S Xyloside primers block proteoglycan glycosylation Cell Fritz & Esko (2001) Methods Mol. Biol. 171:309

  29. OAc OAc OAc O O O O AcO AcO NHAc OAc Gal GlcNAc Naphthalene methanol Types of primers

  30. Metabolic oligosaccharide engineering Labeling enables: • detection • enrichment Dube, D. H.; Bertozzi, C. R. Curr. Opin. Chem. Biol.2003, 7, 616-625

  31. The Staudinger ligation is highly selective and “bioorthogonal”

  32. Many nuclear and cytosolic proteins are transiently modified with b-O-GlcNAc RNApol II Myc p53 Tau • Complete repertoire of O-GlcNAcylated proteins? • Sites of O-GlcNAcylation?

  33. System-wide analysis of b-GlcNAcylated proteins GlcNAz Vocadlo, D. J., et al.PNAS 2003,100, 9116-9121

  34. W. Reutter et al. Unnatural ManNAc analogs are metabolized to unnatural sialic acids in cells Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Science1997, 276, 1125-1128 Luchansky, S. J.; Goon, S.; Bertozzi, C. R. ChemBioChem2004, 5, 371-374

  35. Unnatural sialic acids can serve as PSA chain terminators Mahal, L. K., et al. Science2001, 294, 380-382

  36. The Staudinger ligation can target sialylatedcells with chemical probles Saxon, E.; Bertozzi, C. R. Science2000, 287, 2007-2010

  37. Imaging changes in glycan expression within living animals FLAG • Glycosylation changes associated with disease • Glycosylation changes during development

  38. Carbohydrate- specific receptor Cell surface glycoconjugates Metabolic interconversions Monosaccharide "building blocks" Glycoconjugate assembly Chemical approaches for perturbing cellular glycans Substrates ER/Golgi Cytosol Bertozzi, C. R.; Kiessling, L. L. Science2001, 291, 2357.

  39. “GlycoChip” from Glycominds Glycan arrays can be used to probe lectin/enzyme specificity

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