title: Portable sulphotransferase domain determines sequence specificity of heparan sulphate 3-O-sulphotransferases.
abstract: 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-3(A) to generate N1-ST3(A). Similarly, the N-terminal region of 3-OST-3(A) was fused to the sulphotransferase domain of 3-OST-1 to generate N3(A)-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-3(A), indicating its significantly greater selectivity for substrates that can be 3-O-sulphated to yield such sites. 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-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.
authors.affiliation: Angiogenesis Research Center, Department of Medicine, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
authors.forename: T
authors.initials: T
authors.lastname: Yabe
authors.forename: D
authors.initials: D
authors.lastname: Shukla
authors.forename: P G
authors.initials: PG
authors.lastname: Spear
authors.forename: R D
authors.initials: RD
authors.lastname: Rosenberg
authors.forename: P H
authors.initials: PH
authors.lastname: Seeberger
authors.forename: N W
authors.initials: NW
authors.lastname: Shworak
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells / Q000201:enzymology; D019556:COS Cells / Q000201:enzymology; D002678:Chimera; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006497:Heparan Sulfate; D006561:Herpes Simplex / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D000991:Antithrombins; D017931:DNA Primers; D018076:DNA, Complementary; D006497:Heparan Sulfate; D007527:Isoenzymes; D020033:Protein Isoforms; D011994:Recombinant Proteins; D015238:Sulfotransferases; C021350:heparitin sulfotransferase