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

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

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

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

Examples of chondroitin sulfate proteoglycans

Examples of keratan sulfate proteoglycans Essentials of Glycobiology

Examples of heparan sulfate proteoglycans Essentials 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

Use of Denaturing Chaotropic Agents to Isolate Proteoglycans:

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

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

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

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

Attachment of GAGs to Protein Core:

GAGs are Often Attached at SG Sites:

Chondroitin Sulfate Attachment Sites:

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

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

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

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.

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

Degrades All Chondroitin Sulfates, Dermatan Sulfates and Hyaluronic Acid

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

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

Keratanases Essentials of Glycobiology

Heparinase Specificities

Heparin Fragments on a 20% Acrylamide Gel:

Typical Repeating Disaccharides

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

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

Disaccharides Released by Chondroitinase:

Paper Chromatography of Released Disaccharides

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,).

Attaching a Chromophore for Analysis: Sigma Chemical Company

HPLC separation of CS-derived saturated and unsaturated disaccharides labeled with 2AB FIG. 2. HPLC separation of CS-derived saturated and unsaturated disaccharides labeled with 2AB. CS-derived saturated tetrasaccharides Tetra-C1–C4 were individually digested with chondroitinase AC-II and derivatized with 2AB. The four digests and 2ABderivatized unsaturated CS-disaccharides were mixed and analyzed by HPLC as described in the legend to Fig. 1. The HPLC conditions were the same as those for the experiments shown in Fig. 1.

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

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

GAG Disaccharides from MDCK Cells Heparinase CSase ABC

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

Anion-Exchange Analysis of Linkage Region:

Scheme for Sequencing CS:

FACE Analysis of Disaccharides

FACE Analysis of GAG-Derived Disaccharides:

Identifying the Non-Reducing Ends

Using Mercuric Acetate to ID Reducing Ends:

Using FACE to Analyze Non-Reducing Ends:

Specific Enzymes to Confirm Sulfation:

Specific Enzymes to Confirm Sulfation:

Biosynthesis of Chondroitin Sulfate 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.

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

FT-ICR negative-ion mass spectrum of 5 FT-ICR negative-ion mass spectrum of 5.80-kDa MR fractionby PAGE with 18 isobars and 63 parent ions. Deconvolution CID-FT-ICR-MS/MS spectra of parent-ion m/z = 917.38 fraction. (a) FT-ICR negative-ion mass spectrum of 5.80-kDa MR fraction by PAGE with 18 isobars and 63 parent ions. (b) Deconvolution of spectrum a. (c) CID-FT-ICR-MS/MS spectra of parent-ion m/z = 917.38 (z = 6) and annotated fragment-ions providing sequence with dp27-5-Ser fragmentation pattern assigned from spectrum. Sequencing by CID NATURE CHEMICAL BIOLOGY | VOL 7 | NOVEMBER 2011

Flow Chart for Analysis: Flow chart reads from left to right. The MR (kDa) determined based on PAGE of fractions (blue rectangles represent gel bands) of bikunin in peptidoglycosaminoglycan prepared by continuous elution PAGE is shown. The deconvoluted MS obtained using FT-ICR-MS affords the mass of 3–5 odd and even components (green ovals) observed in each bikunin peptidoglycosaminoglycan fraction is shown. Each MS spectrum showed multiple charge states (z values) shown as red diamonds from which parent ions were selected for MS/MS giving CID fragments by analysis on FT (purple circles) or FT-ICR (brown circles). A shorthand sequence for each chain is shown with a, b, c and d subdomain repeats indicated by numbers (that is, 0.2.0.3 for d = 0, c = 2, b = 0, a = 3). The overall sequence of bikunin chondroitin sulfate-A peptidoglycosaminoglycan shown at the bottom is consistent with all determined sequences. 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:10.1038/nchembio.673

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.