Portable sulphotransferase domain determines sequence specificity of heparan sulphate 3-O-sulphotransferases

3-O-Sulphates are the rarest substituent of heparan sulphate and are theref ore ideally suited to the selective regulation of biological activities. In dividual isoforms of heparan sulphate D-glucosaminyl 3-O-sulphotransferase (3-OST) exhibit sequence -specific action, which creates heparan sulphate s tructures with distinct biological functions. For example. 3-OST-1 preferen tially 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 su lphotransferase domain and a divergent N-terminal region. To localize deter minants of sequence specificity. we conducted domain swaps between cDNA spe cies. The N-terminal region of 3-OST-1 was fused with the sulphotransferase domain of 3-OST-3(A) to generate N1-ST3(A). Similarly, the N-terminal regi on of 3-OST-3, was fused to the sulphotransferase domain of 3-OST-1 to gene rate N3(A)-ST1. Wild-type and chimaeric enzymes were transiently expressed in COS-7 cells and extracts were analysed for selective generation of bindi ng sites for anti-thrombin. 3-OST-1 was 270-fold more efficient at forming a ri thrombin-binding sites than 3-OST-3(A), indicating its significantly g reater selectivity for substrates that can be 3-O-sulphated to yield such s ites. N3(A)-ST1 was as active as 3-OST-1. whereas the activity of N1-ST3(A) , was as low as that of 3-OST-3(A). Analysis of Chinese hamster ovary cell transfectants revealed that only 3-OST-3(A) and N1-ST3(A) generated gD-bind ing sites and conveyed susceptibility to infection by HSV-1. Thus sequence- specific properties of 3-OSTs are defined by a self-contained sulphotransfe rase domain and are not directly influenced by the divergent N-terminal reg ion.