Evolutionary Differences in Glycosaminoglycan Fine Structure Detected by Quantitative Glycan Reductive Isotope Labeling

Abstract

To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes ( 12 C 6 and 13 C 6 ). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.Metazoans make several types of sulfated glycosaminoglycans (GAGs), 2 including keratan sulfate (KS), chondroitin sulfate/dermatan sulfate (CS/DS), and heparan sulfate/heparin (HS). Each type of chain consists of unique disaccharide units. KS consists of galactose (Gal) and GlcNAc ([Gal␤1, 4GlcNAc␤1,3] n ) with variable sulfation at C6 of either sugar. CS/DS assembles as a copolymer of GlcA␤1,3GalNAc␤1,4 and then undergoes various processing reactions, including C5 epimerization of a portion of GlcA units to iduronic acid in DS, O-sulfation at C2 and more rarely at C3 of the uronic acids, and O-sulfation at C4 and C6 of the GalNAc residues (1). HS is the most highly modified GAG, consisting initially of GlcA␤1,4GlcNAc␣1,4 units, which then undergo variable processing by GlcNAc N-deacetylation and N-sulfation, C5 epimerization of some GlcA units to iduronic acid, and O-sulfate addition to C2 of the uronic acids and C6 and more rarely at C3 of the glucosamine units (2). The arrangement of the modified residues along the chain creates binding sites for numerous growth factors, enzymes, and extracellular matrix proteins. The structural variation that can occur makes sulfated GAG chains one of the most complex classes of macromolecules found in nature.GAG fine structure is typically assessed by analyzing the disaccharide composition of an isolated mixture of chains. A number of techniques have been developed to accomplish this task that rely on chemical or enzymatic depolymerization of the chains into their constituent disaccharides, followed by separation via anion exchange chromatography, reversed-phase chromatography with ion pairing agents, or capillary electrophoresis. These techniques separate...

Lacrimal Gland Development and Fgf10-Fgfr2b Signaling Are Controlled by 2-O- and 6-O-sulfated Heparan Sulfate

“…Heparan Sulfate Disaccharide Compositional Analysis-Disaccharide analysis of heparan sulfate extracted from primary MEFs was carried out using procedures previously described (37). Briefly, heparan sulfate was extracted from MEF cell pellets by exhaustive digestion with Pronase (Sigma) in PBS at 37°C for 24 h followed by anion-exchange chromatography (DEAE-Sephacel, GE Healthcare).…”

Lymphatic Endothelial Heparan Sulfate Deficiency Results in Altered Growth Responses to Vascular Endothelial Growth Factor-C (VEGF-C)

“…Heparan sulfate disaccharide analysis was carried out by quantitative liquid chromatography/mass spectrometry (21). Briefly, samples were dried down, and [ 12 C 6 ]aniline (15 l, 165 mol) and 15 l of 1 M NaCNBH 3 (Sigma) freshly prepared in dimethyl sulfoxide/acetic acid (7:3, v/v) were added to each sample.…”

Identification of Chemically Sulfated/desulfated Glycosaminoglycans in Contaminated Heparins and Development of a Simple Assay for the Detection of Most Contaminants in Heparin

“…[15][16][17] Heparin lyase digested samples were labeled with H-aniline or D5-aniline by reductive amination. In contrast, low pH nitrous acid degraded samples were labeled with NaBH 4 or NaBD 4 .…”

“…In these experiments, the products derived from chondroitinase digestion, consisting mostly of disaccharides, were analyzed by NaBH 4 reductive amination with isotope-tagged aniline and liquid chromatography/ mass spectrometry. 15 This technique allows determination of the number of sulfate groups (and their position) on each disaccharide. As shown in Table 2, the chondroitin-like material in G1, G2, and G3 consisted of non-sulfated disaccharides (55%-57% of the total), monosulfated disaccharides (18%-20%) and disulfated disaccharides (24%-27%).…”

“…These results indicate that the DAUA+1S+103 and DAUH+3S+103 structures (ion current intensities are shown in Table 3) were not artifacts of the assay systems but represented chemically modified heparin/ heparan sulfate because such structures were not detected in un-adulterated heparin or in other naturally occurring heparan sulfate by using the same liquid chromatography/mass spectrometry analysis approach. 15 establish a simple heparin quality control assay…”