Upload
jaylynn-tillinghast
View
224
Download
4
Embed Size (px)
Citation preview
-Techniques in Glycobiology-NHLBI CardioPEG – Gerald W.Hart, September 17, 2013
Funded by NHLBI P01HL107153
Analysis ofProteoglycans
&Glycosaminoglyc
ans
2
Proteoglycans consist of a protein core and one or more covalently attached glycosaminoglycan chains
Chapter 16, Figure 2Essentials of Glycobiology
Second Edition
3
Glycosaminoglycans consist of repeating disaccharide units
Chapter 16, Figure 3Essentials of Glycobiology
Second Edition
4
Keratan sulfates contain a sulfated poly-N-acetyllactosamine chain
Chapter 16, Figure 4Essentials of Glycobiology
Second Edition
5
Examples of chondroitin sulfate proteoglycans
6
Examples of keratan sulfate proteoglycans
Essentials of Glycobiology
Examples of heparan sulfate proteoglycans
7Essentials of Glycobiology
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
Use of Denaturing Chaotropic Agents to Isolate Proteoglycans:
9
Isolation of Intact Proteoglycans
10Proteoglycan ProtocolsEdited by Renato V. Iozzo, MD
35SO4 + 3H-glucosamine
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 DigestionBeta-Elimination
GAG size fractionation – TSK4000, HPLC, Sephadex G200, Superose CL-6B
11
12Proteoglycan ProtocolsEdited by Renato V. Iozzo, MD
Negative Charge Allows Ready Separation from Other Glycans
PGsGPs
Size Fractionation of Proteoglycans
13Proteoglycan ProtocolsEdited by Renato V. Iozzo, MD
Attachment of GAGs to Protein Core:
14
GAGs are Often Attached at SG Sites:
15
Chondroitin Sulfate Attachment Sites:
16
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
GAG Structure – 3 Regions: Linkage, Repeat, Non-Reducing Terminus:
18
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-GlycansLinkage of oligosaccharidesO-Glycans in beta-eliminated GAGS
19
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.
20
21
Characterization of Glycosaminoglycans:
Bacterial EliminasesAre Powerful Tools:
1. Sequential Degradation Followed by Gel Filtration.
2. Other Separation Methods.
22Degrades All Chondroitin Sulfates, Dermatan Sulfates and
Hyaluronic Acid
23
Digests All Types of Chondroitin Sulfates and Hyaluronic Acids, but Not Digest Hyaluronic Acids.
24
25
Hyaluronidases (eg. testicular; a hydrolyase)
Also DegradesChondroitin sulfates
26
27
Keratanases
28
Essentials of Glycobiology
Heparinase Specificities
29
Heparin Fragments on a 20%
Acrylamide Gel:
30
Typical Repeating Disaccharides
31
Nitrous Acid Degradation of Heparan Sulfate & Heparin
32
Nat. Prod. Rep. , 2002, 19, 312-331
Analytical Methods for GAGRepeat Disaccharides
Paper ChromatographyThin Layer ChromatographyHPLC MethodsCapillary ElectrophoresisFluorophore-Assisted
Carbohydrate Electrophoresis (FACE)
33
Disaccharides Released byChondroitinase:
34
Paper Chromatography of Released Disaccharides
35
36
Thin-Layer Chromatography of Released Disaccharides
Silica Gel 60 TLC aluminum plate and developed with a solventsystem consisting of n -butanol/formic acid/water (4:8:1,).
Attaching a Chromophore for Analysis:
37
Sigma Chemical Company
38
HPLC separation of CS-derived saturated and unsaturateddisaccharides labeled with 2AB
39
Separation of AMAC-Labeled Disaccharides by RP-HPLC:
HS
CS
40
Disaccharides from Rat Liver GAGs-SAX-HPLC
HeparinasesCS-ABCase
41
GAG Disaccharides from MDCK Cells
Heparinase
CSase ABC
2-aminobenzamide (2AB) labeled Disaccharides on Anion Exchange Columns:
42
STDs Brain
CartilageSkin
Anion-Exchange Analysis of Linkage Region:
43
Linkage Region
Scheme for Sequencing CS:
44
FACE Analysis of Disaccharides
45
46
FACE Analysis of GAG-Derived Disaccharides:
Identifying the Non-Reducing Ends
47
Using Mercuric Acetate to IDReducing Ends:
48
Using FACE to Analyze Non-Reducing Ends:
49
50
Specific Enzymes to Confirm Sulfation:
Specific Enzymes to Confirm Sulfation:
51
Biosynthesis of Chondroitin Sulfate
52
NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011
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
MS/MS FT-ICR-MS CS GAG
54
NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011
Complete conversion to Na salt
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 = 917.38
Sequencing by CID
56The proteoglycan bikunin has a defined sequenceMellisa Ly, Franklin E Leach III, Tatiana N Laremore, Toshihiko Toida, I Jonathan Amster & Robert J LinhardtNature Chemical Biology 7, 827–833 (2011) doi:10.1038/nchembio.673
Flow Chart for Analysis:
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