Structure of a heparan sulphate oligosaccharide that binds to basic fibroblast growth factor

Abstract

Binding of basic fibroblast growth factor (bFGF) to the extracellular matrix of cultured bovine aorta smooth muscle cells is likely to be mediated via heparan sulphate, since not only exogenous addition of heparan sulphate to the culture medium but also pretreatment of the cells with heparitinase (but not chondroitinase ABC) resulted in loss of binding. Comparison of the affinity of bFGF to various glycosaminoglycan-conjugated gels showed a direct and specific binding of bFGF to heparan sulphate. Heparan sulphate also bound to a bFGF affinity gel. However, the proportion of heparan sulphate bound varied depending on the source of the HS (more than 90% and 45% with pig aorta heparan sulphate and mouse EHS tumour heparan sulphate respectively). The bound heparan sulphate had the ability to protect bFGF from proteolytic digestion, but the unbound heparan sulphate did not. The results suggest the presence in the bound heparan sulphate of a specific structure involved in binding. Limited digestion with heparitinase I of porcine aorta heparan sulphate yielded 13% oligosaccharides bound to the gel, of which the smallest were octasaccharides. Analysis of a hexadecasaccharide fraction which was obtained at the highest yield among the bound oligosaccharides was performed by h.p.l.c. of the deamination products obtained with nitrous acid and the unsaturated disaccharide products formed by heparitinase digestion. Comparison of the disaccharide unit compositions exhibited a marked difference in IdoA(2SO4)GlcNSO3 and IdoA(2SO4)GlcNSO3(6SO4) units between the bound and unbound hexadecasaccharides. The amounts measured were 3 mol and 1 mol per mol of the former and 0.4 mol and 0.6 mol per mol of the latter. It is likely that the binding of bFGF to heparan sulphate may require the domain structure of the heparan sulphate to be composed of clustering IdoA(2SO4)-GlcNSO3 units.

Drosophila Heparan Sulfate 6-O-Sulfotransferase (dHS6ST) Gene

“…We measured 35 S incorporation activities into various glycosaminoglycans of the purified dHS6ST under those condition. Analysis of the reaction products derived from CDSNS-heparin and heparan sulfate (from a mouse EHS tumor) by expressed enzymes was performed as described previously (35,36).…”

Regulation of Heparan Sulfate 6-O-Sulfation by β-Secretase Activity

“…For comparison, the secretion of the stable HS6ST1-GFP transfectant was set to 100%. Total GFP fluorescence was as follows: HS6ST1-GFP, 22 not increase (Fig. 3A, lower left).…”

“…The precipitates were dissolved in water, and portions of the solutions were treated at 37°C for 3 h with a mixture of 1 milliunit of heparitinase I, 2 milliunits of heparitinase II, and 1 milliunit of heparitinase III in 50 l of 50 mM Tris-HCl buffer, pH 7.2, containing 1 mM CaCl 2 and 4 g of bovine serum albumin. After the digests were filtered with Ultrafree-MC (5,000 molecular weight limit; Millipore Corp., Bedford, MA), unsaturated disaccharides in the filtrates were separated by a silicabased polyamine column as described previously (22). The radioactivity of the fraction was determined by using the LS 6500 scintillation counter (Beckman Coulter, Fullerton, CA).…”

Combined strong anion-exchange HPLC and PAGE approach for the purification of heparan sulphate oligosaccharides

“…The S-domains of the HS chains are involved in mediating both recognition and activation of various HS-binding proteins, such as basic fibroblast growth factor [14][15][16][17], hepatocyte growth factor [18], platelet-derived growth factor [19], herpes simplex gC glycoprotein [20] and platelet factor 4 [21]. The binding of Sdomains to proteins has been shown to be specific [6,7].…”