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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 Lecture 41 Carolyn R. Bertozzi UC Berkeley
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.
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
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
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
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
Glycosyltransferase inhibitors based on substrates/transition states
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
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
Consensus sequence None Nothing general Affinity reagents Small molecule inhibitors None Enzymes for cleavage Nothing general Challenges in studying O-linked glycosylation Conserved core
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
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
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
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)
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
Glycosyltransferases are residents of the Golgi compartment cis medial trans TGN
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
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
Rapamycin mediates association of FKBP and FRB Rapamycin J. Liang, J. Choi, & J. Clardy. Acta Cryst. (1999) D55, 745-752
{ FucT7 Fucosyltransferase 7 is involved in selectinligand biosynthesis Leukocyte L-Selectin Endothelial cell
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)
FucT7 activity in CHO cells can be monitored by the expression of sialyl Lewis x Sialyl Lewis x (detected with mAb HECA-452)
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
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.
• 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
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
OAc OAc OAc O O O O AcO AcO NHAc OAc Gal GlcNAc Naphthalene methanol Types of primers
Metabolic oligosaccharide engineering Labeling enables: • detection • enrichment Dube, D. H.; Bertozzi, C. R. Curr. Opin. Chem. Biol.2003, 7, 616-625
The Staudinger ligation is highly selective and “bioorthogonal”
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?
System-wide analysis of b-GlcNAcylated proteins GlcNAz Vocadlo, D. J., et al.PNAS 2003,100, 9116-9121
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
Unnatural sialic acids can serve as PSA chain terminators Mahal, L. K., et al. Science2001, 294, 380-382
The Staudinger ligation can target sialylatedcells with chemical probles Saxon, E.; Bertozzi, C. R. Science2000, 287, 2007-2010
Imaging changes in glycan expression within living animals FLAG • Glycosylation changes associated with disease • Glycosylation changes during development
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.
“GlycoChip” from Glycominds Glycan arrays can be used to probe lectin/enzyme specificity