Comparative glycomics of connective tissue glycosaminoglycans

Abstract

Homeostasis of connective joint tissues depends on the maintenance of an extracellular matrix, consisting of an integrated assembly of collagens, glycoproteins, proteoglycans, and glycosaminoglycans (GAGs). Isomeric chondroitin sulfate (CS) glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work profiles the CS epitopes expressed by different joint tissues as a function of age and osteoarthritis. GAGs were extracted from joint tissues (cartilage, tendon, ligment, muscle, and synovium) and partially depolymerized using chondroitinase enzymes. The oligosaccharide products were differentially stable isotope labeled by reductive amination using 2-anthranilic acid-d(0) or -d(4) and subjected to amide-hydrophilic interaction chromatography (HILIC) online LC-MS/MS. The analysis presented herein enables simultaneous profiling of the expression of nonreducing end, linker region, and Delta-unsaturated interior oligosaccharide domains of the CS chains among the different joint tissues. The results provide important new information on the changes to the expression of CS GAG chains during disease and development.

Figure 1

A. Base peak chromatogram (100–800 m/z) of 30% chondroitin lyase depolymerized CS/DS from 5-μg d₄-2AA-juvenile bovine cartilage mixed with 1-μg d₀-2AA-CSA internal oligosaccharide standard. GAG oligosaccharide chains ranging from disaccharide to dodecasaccharide elute from 15 to 55 minutes. Oligosaccharide compositions are given as (HexA, GalNAc, SO₃) (X,Y,Z), with 4,5-unsaturation shown as Δ. B. The average mass spectrum of all eluted oligosaccharides in the sample mixture. Label of tris indicates reductive amination product with tris buffer.

Figure 2

MS percent total ion abundances of all oligosaccharide chains present in all juvenile bovine and adult human cartilage samples. Oligosaccharide compositions are given as (HexA, GalNAc, SO₃) (X,Y,Z), with Δ indicating 4,5-unsaturation of the non-reducing terminal HexA residue. Tetrasaccharide linker compositions are given as (Xyl, Gal, HexA, SO₃) (V,W,X,Z). Hexasaccharide linker compositions are given as (Xyl, Gal, HexA, GalNAc, SO₃) (V,W,X,Y,Z).

Figure 3

A. The percent total ion abundance of all product ions produced in the tandem mass spectra of Δ(2,2,2) B. The percent total ion abundance of all product ions produced in the tandem mass spectra of Δ(3,3,3) C. The percent total ion abundance of all product ions produced in the tandem mass spectra of Δ(4,4,4). X, Y and [M-H-S0₃] ions correspond to cartilage product ions containing the reducing end and therefore the d₄-2-anthranilic acid tag. B and C ions correspond to the non-reducing end, which does not contain the stable isotope tag, and therefore contain a mixture of cartilage and internal standard. The star denotes product ions that have a statistical difference in ion abundance between the healthy and diseased tissues. The key shown in graph A is applicable to each of the three graphs.

Figure 4

MS percent total ion abundances of all oligosaccharide chains present in all connective tissue samples from juvenile bovine muscle, ligament, tendon and synovium. Linker region oligosaccharide compositions are expanded in order to see lower abundance structures. Oligosaccharide compositions are given as (HexA, GalNAc, SO₃) (X,Y,Z), with Δindicating 4,5-unsaturation of the non-reducing terminal HexA residue. Tetrasaccharide linker oligosaccharides compositions are given as (Xyl, Gal, HexA, SO₃) (V,W,X,Z). Hexasaccharide linker oligosaccharide compositions are given as (Xyl, Gal, HexA, GalNAc, SO₃) (V,W,X,Y,Z). Cartilage has been added to the connective tissue graphs as a point of reference.

Scheme I

The oligosaccharide compositions of eighteen different structures found in connective tissue samples by HILIC-amide LC/MS. The compositions include Δ-unsaturated internal oligosaccharides, over-/under-sulfated Δ-unsaturated oligosaccharides, non-reducing end saturated structures of varying chain length and linker region structures. Oligosaccharide compositions are given as (HexA, GalNAc, SO₃) (X,Y,Z), with 4,5-unsaturation shown as Δ. L₄ represents the tetrasaccharide linker and L₆ represents the hexasaccharide linker oligosaccharide. Internal hexuronic acids may be either glucuronic acid or iduronic acid. Sulfates residing on the galactose and N-acetylgalactosamine have been arbitrarily placed. Sulfates on the N-acetylgalactosamine may be positioned on either the 4- or 6-position. The second sulfate on Δ(2,2,3) may be positioned on either of the two N-acetylgalactosamines.