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-Techniques in Glycobiology -

Analysis of Proteoglycans & Glycosaminoglycans. -Techniques in Glycobiology -. NHLBI CardioPEG – Gerald W.Hart , September 17, 2013 Funded by NHLBI P01HL107153. Proteoglycans consist of a protein core and one or more covalently attached glycosaminoglycan chains.

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-Techniques in Glycobiology -

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

  2. Proteoglycans consist of a protein core and one or more covalently attached glycosaminoglycan chains Essentials of Glycobiology Chapter 16, Figure 2 Second Edition

  3. Glycosaminoglycans consist of repeating disaccharide units Essentials of Glycobiology Chapter 16, Figure 3 Second Edition

  4. Keratan sulfates contain a sulfated poly-N-acetyllactosamine chain Essentials of Glycobiology Chapter 16, Figure 4 Second Edition

  5. Examples of chondroitin sulfate proteoglycans

  6. Examples of keratan sulfate proteoglycans Essentials of Glycobiology

  7. Examples of heparan sulfate proteoglycans Essentials of Glycobiology

  8. Proteoglycan Analysis • Isolation and Analysis of Intact Proteoglycans • Identification of Core Protein – Sequence. • Site-Mapping of Proteoglycans & Other PTMs • Isolation & Characterization of Glycosaminoglycans • Digestions to Produce disaccharide repeat • Determining Repeat Composition • Sulfation • Non-reducing terminus • Sequencing of GAGs

  9. Use of Denaturing Chaotropic Agents to Isolate Proteoglycans:

  10. 35SO4 + 3H-glucosamine Isolation of Intact Proteoglycans Proteoglycan Protocols Edited by Renato V. Iozzo, MD

  11. Typical Work Flow - Proteoglycans • Extraction of Proteoglycans – Typically 4M Urea or 6M GuHCl –Strong denaturing/chaotropic agents. • Membrane bound PGs require Detergents • Ion-Exchange -DEAE-Sephacel or similar anion exchange enrichment. – High negative charge. • Size Exclusion Chromatography – Typically Sepharose 4B • Analysis of GAG chains after release – • Protease Digestion • Beta-Elimination • GAG size fractionation – TSK4000, HPLC, Sephadex G200, Superose CL-6B

  12. Negative Charge Allows Ready Separation from Other Glycans GPs PGs Proteoglycan Protocols Edited by Renato V. Iozzo, MD

  13. Size Fractionation of Proteoglycans Proteoglycan Protocols Edited by Renato V. Iozzo, MD

  14. Attachment of GAGs to Protein Core:

  15. GAGs are Often Attached at SG Sites:

  16. Chondroitin Sulfate Attachment Sites:

  17. Biochemical Site Mapping of GAGs • Similar Approaches as Other O-Glycans eg. • Beta-Elimination/Michael Addition • MS/MS using ETD on PGs with GAGs Truncated

  18. GAG Structure – 3 Regions: Linkage, Repeat, Non-Reducing Terminus:

  19. Analysis of Glycosaminoglycans • Release from PGs – Protease, Beta-Elimination • Lyases & Hydrolases – Fragment GAGs • Disaccharide Compostion Analysis • Sulfation Sites • Non-Reducing Terminus • Mercuric acetate elimination of unsaturated bond containing disaccharides reveals non-reducing Terminus • Presence of Classical N- & O-Glycans • Linkage of oligosaccharides • O-Glycans in beta-eliminated GAGS

  20. GAG Degrading Enzymes:Hydrolases & Eliminases: • Hydrolase – Catalyzes Hydrolysis i.e. Addition of water across a chemical bond. A-B + H2O  A-OH + B-H Examples: testicular Hyase; endo-b-galactosidase • Eliminase – Catalyzes the removal of H2O from a chemical bond. A-B  A-OH + dB + H2O Examples: chondroitinase ABC, HS lyases, Strept. Hyaluronidase.

  21. Characterization of Glycosaminoglycans: Bacterial Eliminases Are Powerful Tools: Sequential Degradation Followed by Gel Filtration. 2. Other Separation Methods.

  22. Degrades All Chondroitin Sulfates, Dermatan Sulfates and Hyaluronic Acid

  23. Digests All Types of Chondroitin Sulfates and Hyaluronic Acids, but Not Digest Hyaluronic Acids.

  24. Hyaluronidases (eg. testicular; a hydrolyase) Also Degrades Chondroitin sulfates

  25. Keratanases Essentials of Glycobiology

  26. Heparinase Specificities

  27. Heparin Fragments on a 20% Acrylamide Gel:

  28. Typical Repeating Disaccharides

  29. Nitrous Acid Degradation of Heparan Sulfate & Heparin Nat. Prod. Rep. , 2002, 19, 312-331

  30. Analytical Methods for GAGRepeat Disaccharides • Paper Chromatography • Thin Layer Chromatography • HPLC Methods • Capillary Electrophoresis • Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)

  31. Disaccharides Released byChondroitinase:

  32. Paper Chromatography of Released Disaccharides

  33. Thin-Layer Chromatography of Released Disaccharides Silica Gel 60 TLC aluminum plate and developed with a solvent system consisting of n -butanol/formic acid/water (4:8:1,).

  34. Attaching a Chromophore for Analysis: Sigma Chemical Company

  35. HPLC separation of CS-derived saturated and unsaturated disaccharides labeled with 2AB

  36. Separation of AMAC-Labeled Disaccharides by RP-HPLC: HS CS

  37. Disaccharides from Rat Liver GAGs-SAX-HPLC Heparinases CS-ABCase

  38. GAG Disaccharides from MDCK Cells Heparinase CSase ABC

  39. STDs 2-aminobenzamide (2AB) labeled Disaccharides on Anion Exchange Columns: Brain Cartilage Skin

  40. Anion-Exchange Analysis of Linkage Region: Linkage Region

  41. Scheme for Sequencing CS:

  42. FACE Analysis of Disaccharides

  43. FACE Analysis of GAG-Derived Disaccharides:

  44. Identifying the Non-Reducing Ends

  45. Using Mercuric Acetate to IDReducing Ends:

  46. Using FACE to Analyze Non-Reducing Ends:

  47. Specific Enzymes to Confirm Sulfation:

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