3-O-Sulphates are the rarest substituent of heparan sulphate and are therefore ideally suited to the selective regulation of biological activities. Individual isoforms of heparan sulphate d-glucosaminyl 3-O-sulphotransferase (3-OST) exhibit sequence-specific action, which creates heparan sulphate structures with distinct biological functions. For example, 3-OST-1 preferentially generates binding sites for anti-thrombin, whereas 3-OST-3 isoforms create binding sites for the gD envelope protein of herpes simplex virus 1 (HSV-1), which enables viral entry. 3-OST enzymes comprise a presumptive sulphotransferase domain and a divergent N-terminal region. To localize determinants of sequence specificity, we conducted domain swaps between cDNA species. The N-terminal region of 3-OST-1 was fused with the sulphotransferase domain of 3-OST-3A to generate N1-ST3A. Similarly, the N-terminal region of 3-OST-3A was fused to the sulphotransferase domain of 3-OST-1 to generate N3A-ST1. Wild-type and chimaeric enzymes were transiently expressed in COS-7 cells and extracts were analysed for selective generation of binding sites for anti-thrombin. 3-OST-1 was 270-fold more efficient at forming anti-thrombin-binding sites than 3-OST-3A, indicating its significantly greater selectivity for substrates that can be 3-O-sulphated to yield such sites. N3A-ST1 was as active as 3-OST-1, whereas the activity of N1-ST3A was as low as that of 3-OST-3A. Analysis of Chinese hamster ovary cell transfectants revealed that only 3-OST-3A and N1-ST3A generated gD-binding sites and conveyed susceptibility to infection by HSV-1. Thus sequence-specific properties of 3-OSTs are defined by a self-contained sulphotransferase domain and are not directly influenced by the divergent N-terminal region.

Abbreviations used: 3-OST, heparan sulphate d-glucosaminyl 3-O-sulphotransferase; AT, anti-thrombin; CHO, Chinese hamster ovary; FBS, fetal bovine serum; GlcNS, N-sulpho-d-glucosamine; HS, heparan sulphate; HSact, HS with AT-binding sites; HSV-1, herpes simplex virus 1; IdoA2S, l-iduronic acid 2-O-sulphate; NDST, HS N-deacetylase/N-sulphotransferase; PAPS, adenosine 3′-phosphate 5′-phosphosulphate.

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Author notes


Present address: Department of Molecular Microbiology and Immunology, University of Missouri-Columbia, M610 G, Medical Science Building, 1 Hospital Drive, Columbia, MO 65211, U.S.A.