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-Techniques in Glycobiology -

Analysis of GPI-Anchors. -Techniques in Glycobiology -. NHLBI CardioPEG – Gerald W.Hart , September 17, 2013 Funded by NHLBI P01HL107153. VSG’s Critical Role in History of GPI-Anchor Analysis:. T. Brucei 10 7 VSG per Cell!.

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-Techniques in Glycobiology -

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  1. Analysis of GPI-Anchors -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, September 17, 2013 Funded by NHLBI P01HL107153

  2. VSG’s Critical Role in History of GPI-Anchor Analysis:

  3. T. Brucei 107 VSG per Cell!

  4. In Blood Stream Trypansomes Defeat the Immune System by Switching Their VSG Coats!

  5. Tightly Packed VSG Shields the Parasite From the Immune System: Ferguson, M..A.J. Journal of Cell Science 112, 2799-2809 (1999)

  6. Relationship of GPI-Anchors to Phosphatidylinositol Amide Linkage to COOH Terminus of Protein. Ram Vishwakarma CSIR-Indian Institute of Integrative Medicine, Jammu National Institute of Immunology, New Delhi PiramalLife Sciences Ltd, Mumbai

  7. Non-Acetylated GlcN

  8. Structure of Triton-X114 n=7 or 8

  9. Triton-X114 Partitioning is a Simple & Powerful Method for Isolation of GPI-Anchor Proteins.

  10. Example of the Use of TX-114 Partitioning for GPI-Anchored Proteins:

  11. Tot P insol. Aq pellet final Det phase TX-114 is a Powerful Tool:

  12. SDS HepesHepesHepes +NP40 5mM Zn Isolation in SDS conserves Anchor, but short incubation endogenous GPI-PLC Releases sVSG 3H-myr Coomassie T S P T S P T S P

  13. TCA PPt Cells Extract pellet with Chloroform:Methanol Add NaCl soln. to form 2 phases mfVSG in Aqueous Wcells CM IfacialAqph Wcells TCA pptCM:Met TCA of Supnt of 3 Purification of mfVSG and sVSG: Incubate tyrps in buffer with organic to lyse cells

  14. sVSG MfVSG has ONLY Myristic Acid: mfVSG

  15. Separation of mfVSG and sVSG on 7.5% SDS-PAGE mfVSG Mix sVSG

  16. GPI-Anchor Biosynthetic Pathway Was Mostly Elucidated by Thin-Layer Chromatography and Pulse-Chase Labelling: Studying GPI Biosynthesis invitro thin layer chromatography F 30 °C O add solvents cell membranes spin salts,buffers evaporate radiolabeled Sugar donor O F From Varki

  17. Radiolabeling & TLC Were Used to Elucidate the GPI-Anchor Biosynthetic Pathway:

  18. Incorporation of 3H-Glucosamine UDP-[3H]GlcNAc UDP-[3H]GlcNAc In Vivo Cell, Vol. 56, 793400, March 10, 1989,

  19. Pulse with UDP-3H-GlcNAcChase UDP-GlcNAc & GDP-Mannose 3H -Myrstate • In Vivo • + • PI-PLC Pulse-Chase Labeling Reveals Intermediates in Pathway: In vitro UDP-GlcNAc + GDP-Man No UDP-GlcNAc Early Studies Suggested Lipid Remodeling of GPI-Anchor Treated with GPI-PLC No GDP-Man MPD Inositol Acylated A’=other F.A. GPI A=dimyrisoyl GPI Lyso-Lipid Remodeling Intermediate Cell, Vol. 56, 793400, March 10, 1989,

  20. Characterization of GPI-Anchor Intermediates: Cell, Vol. 56, 793400, March 10, 1989,

  21. Glycolipids A and A’ Have The Same Glycan Structure: Intermediates After HONO Treatment Leaves Glycan with AHM At Reducing Terminus. Gel Filtration – P4 Dionex HIPC-ASG Cell, Vol. 56, 793400, March 10, 1989,

  22. Biosynthetic Pathway of VSG GPI Insect Form of Parasite Blood Stream Form FEBS Letters 584 (2010) 1670–1677

  23. Schematic Structure & Cleavage Sites of GPI-Anchors:

  24. Flow Chart For GPI-Anchor Protein Analysis:

  25. Chemical and enzymatic reactions of GPI anchors Essentials of Glycobiology Chapter 11, Figure 3 Second Edition

  26. Ferguson et al. Partial Structure Elucidation of GPI-Anchor: JBCVol. 260, pp. 14547-14555 1985 1 • Treatment with HONO releases intact PI. • Treatment of sVSG (released by PI-PLC) with Pronase yields glycopeptide. • GP insensitive to alkaline phos. & yields myo-inositol 1-P on HONO treatment. • Mild acid renders sensitive to alkphos. • Subsequent HONO yields myoinositol • HONO yields AHM. 2 3,4 5 6

  27. Linkages Were Determined by Methylation Analysis: Glycan  Methylate all Free Hydroxyl Groups (Ether Linkages, Acid Stable)Acid HydrolyzeReduce and Per-Acetylate (Alditol Acetates)  GC Analysis

  28. Size of the Glycan Moiety And Structure Was Estimated by Gel Filtration in Combination With Specific Degradation (Easily Resolve a Hexose Difference):

  29. NMR Was Used to Determine the Glycan Structure of the GPI:

  30. Proteomics/Glycomics of GPI-Proteins Molecular & Cellular Proteomics 2: 1261–1270, 2003.

  31. >CRD Ab Released to Sol by PLC Isolation of GPI-anchored Proteins: Molecular & Cellular Proteomics 2: 1261–1270, 2003.

  32. Example of MS Data: Band 1 Molecular & Cellular Proteomics 2: 1261–1270, 2003.

  33. MS/MS Identification: Molecular & Cellular Proteomics 2: 1261–1270, 2003.

  34. Algorithms for Predicting GPI-Anchor Attachment:

  35. Conclusions: • GPI-Anchor’s Unique Structures Require Unique Approaches. • PI-PLC release from membranes is diagnostic for GPI-Anchors • Triton-X114 Partitioning is a Great Method to Start Purification of GPI-Anchored Proteins • Reductive Labeling of Anhydromannose after nitrous acid treatment is a powerful tool. • Aqueous HF releases GPI from protein. • Sizing and sequential use of glycosidases use same methods as other oligosaccharides. • Consensus sequence for predicting GPIs.

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