Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations

Presentation on theme: "-Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, September 17, 2013 Funded by NHLBI P01HL107153 Analysis of Proteoglycans & Glycosaminoglycans."— Presentation transcript:

1 -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, September 17, 2013 Funded by NHLBI P01HL Analysis of Proteoglycans & Glycosaminoglycans

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

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

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

5 5 Examples of chondroitin sulfate proteoglycans

6 6 Examples of keratan sulfate proteoglycans Essentials of Glycobiology

7 Examples of heparan sulfate proteoglycans 7 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 8

9 Use of Denaturing Chaotropic Agents to Isolate Proteoglycans: 9

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

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 11

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

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

14 Attachment of GAGs to Protein Core: 14

15 GAGs are Often Attached at SG Sites: 15

16 Chondroitin Sulfate Attachment Sites: 16

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 17

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

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 19

20 GAG Degrading Enzymes: Hydrolases & Eliminases:  Hydrolase – Catalyzes Hydrolysis i.e. Addition of water across a chemical bond. A-B + H 2 O  A-OH + B-H Examples: testicular Hyase; endo-  -galactosidase  Eliminase – Catalyzes the removal of H 2 O from a chemical bond. A-B  A-OH + dB + H 2 O Examples: chondroitinase ABC, HS lyases, Strept. Hyaluronidase. 20

21 21 Characterization of Glycosaminoglycans: Bacterial Eliminases Are Powerful Tools: 1.Sequential Degradation Followed by Gel Filtration. 2. Other Separation Methods.

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

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

24 24

25 25 Hyaluronidases (eg. testicular; a hydrolyase) Also Degrades Chondroitin sulfates

26 26

27 27

28 Keratanases 28 Essentials of Glycobiology

29 Heparinase Specificities 29

30 Heparin Fragments on a 20% Acrylamide Gel: 30

31 Typical Repeating Disaccharides 31

32 Nitrous Acid Degradation of Heparan Sulfate & Heparin 32 Nat. Prod. Rep., 2002, 19,

33 Analytical Methods for GAG Repeat Disaccharides  Paper Chromatography  Thin Layer Chromatography  HPLC Methods  Capillary Electrophoresis  Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) 33

34 Disaccharides Released by Chondroitinase: 34

35 Paper Chromatography of Released Disaccharides 35

36 36 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,).

37 Attaching a Chromophore for Analysis: 37 Sigma Chemical Company

38 38 HPLC separation of CS-derived saturated and unsaturated disaccharides labeled with 2AB

39 39 Separation of AMAC-Labeled Disaccharides by RP-HPLC: HS CS

40 40 Disaccharides from Rat Liver GAGs-SAX-HPLC Heparinases CS-ABCase

41 41 GAG Disaccharides from MDCK Cells Heparinase CSase ABC

42 2-aminobenzamide (2AB) labeled Disaccharides on Anion Exchange Columns: 42 STDs Brain Cartilage Skin

43 Anion-Exchange Analysis of Linkage Region: 43 Linkage Region

44 Scheme for Sequencing CS: 44

45 FACE Analysis of Disaccharides 45

46 46 FACE Analysis of GAG-Derived Disaccharides:

47 Identifying the Non-Reducing Ends 47

48 Using Mercuric Acetate to ID Reducing Ends: 48

49 Using FACE to Analyze Non-Reducing Ends: 49

50 50 Specific Enzymes to Confirm Sulfation:

51 51

52 Biosynthesis of Chondroitin Sulfate 52 NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011

53 Difficulties in Sequencing GAGs:  Lack of sufficient quantities of pure proteoglycans,  the multiple sequences possible for the multiple GAG chains often present on a single core protein,  the difficulties in purifying a single GAG chain for sequencing  difficulties in determining GAG sequence.  Why Bikunin:  Bikunin is a member of the kunin family of serine protease inhibitors13–15, is a therapeutically relevant proteoglycan that is used in Japan as a drug for the treatment of acute pancreatitis. Thus, bikunin is available at a high level of purity in multimilligram quantities.  Bikunin has the simplest chemical structure of any proteoglycan, with a single site for O-linked modification by a GAG chain, located at Ser10 in its 16-kDa core protein.  The protein component of bikunin is well characterized, but its GAG chain structure is heterogeneous and has received less attention because of the technical difficulties associated with GAG analysis.  GAG chain is quite short, it is very heterogeneous in size and composition, with 27–39 saccharide residues and a molecular mass (MR) ranging from 5,505 Da to 7,102 Da23.  In addition, enzymatic analysis shows that the bikunin GAG chains contain single- uronic-acid stereochemistry (glucuronic acid), sulfo groups at only the 4 position of its galactosamine residue and no N-sulfo group or N-acetyl group variability, which is common in the GAG chains of the more structurally complex members of the heparan sulfate proteoglycan family. 53

54 MS/MS FT-ICR-MS CS GAG 54 NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011 Complete conversion to Na salt

55 FT-ICR negative-ion mass spectrum of 5.80-kDa MR fractionby PAGE with 18 isobars and 63 parent ions. 55 NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011 Deconvolution CID-FT-ICR-MS/MS spectra of parent-ion m/z = Sequencing by CID

56 56 The proteoglycan bikunin has a defined sequence Mellisa Ly, Franklin E Leach III, Tatiana N Laremore, Toshihiko Toida, I Jonathan Amster & Robert J Linhardt Nature Chemical Biology 7, 827–833 (2011) doi: /nchembio.673 Flow Chart for Analysis:

57 Analysis of Proteoglycans  Advances in Molecular Biology Have Allowed a Detailed Understanding of Core Proteins.  Site Mapping is Similar to other O-Glycans.  GAG Analysis is Greatly Facilitated by the High Specificity of Bacterial Lyases.  Detailed Sequencing of GAGs is still Very Difficult.  Current Technology is NOT Capable of Defining the molecular Species of a Proteoglycan = Information Content.  Recent Developments in Mass Spectrometry are Showing Promise. 57

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

Similar presentations

Ads by Google