Generating heparan sulfate saccharide libraries for glycomics applications

Abstract

Natural and semi-synthetic heparan sulfate (HS) saccharide libraries are a valuable resource for investigating HS structure-function relationships, enabling high-throughput glycomics studies. Owing to the difficulty of chemical or in vitro enzymatic synthesis of HS saccharides, the structural diversity displayed in saccharides from tissue or cell sources cannot be readily accessed. In contrast, saccharide libraries can be generated by partial digestion of tissue-derived HS polysaccharide chains and chromatographic fractionation of the resulting saccharide mixtures. Fractionation is initially on the basis of hydrodynamic volume, using size exclusion chromatography. Further fractionation, on the basis of charge using strong anion exchange, can subsequently be applied. Desalting and sample concentration follows each fractionation step. Chromatographic fractions are generated that contain purified, or partially purified, saccharides. Here we describe a comprehensive protocol for generation of structurally diverse natural saccharide libraries from HS variants that is fast (approximately 3 weeks) and reproducible.

The Solution Structure of Heparan Sulfate Differs from That of Heparin

“…Ϫ1 at 232 nm was used for HS experiments (28). PAGE of HS Fragments-The HS fragments were analyzed by PAGE to determine the level of purity of each one according to a previously described method (29).…”

The effect of the decoy molecule PA401 on CXCL8 levels in bronchoalveolar lavage fluid of patients with cystic fibrosis

“…1A), but in contrast CXCL8 present in CF BALF (n=13) ran as a high molecular weight smear, most likely representing a GAG complexed form of the chemokine as previously described (Reeves et al, 2011b). Alcian blue, the most widely used cationic dye for demonstration of GAGs, and Azure A for sulphated material (Powell et al, 2010), confirmed the presence of GAGs in all CF BALF samples tested (Fig. 1B).…”

“…Gels were run at 130 V for 1.5 h and were stained with Alcian Blue Solution (Merck Millipore) or Azure A chloride stain overnight (Powell et al, 2010) and then destained with ultra pure water.…”

Diversification of the Structural Determinants of Fibroblast Growth Factor-Heparin Interactions

“…Porcine mucosal heparan sulfate, hyaluronic acid (HA), and chondroitin sulfate (CS C) were from Sigma; dermatan sulfate (DS) was from Iduron (Manchester, UK). Heparin oligosaccharides of degrees of polymerization (DP) DP2, DP4, DP6, DP8, DP10, and DP12 were produced, as described previously (18). A range of systematically modified heparin derivatives with different repeating units (D1-9) were prepared as described previously (Table 1) (19).…”