Abstract
Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine residue of heparan sulfate (HS) to form 3-O-sulfated HS. The 3-O-sulfated glucosamine residue contributes to two important biological functions of HS: binding to antithrombin and thereby carrying anticoagulant activity, and binding to herpes simplex viral envelope glycoprotein D to serve as an entry receptor for herpes simplex virus 1. A total of five HS 3-O-sulfotransferase isoforms were reported previously. Here we report the isolation and characterization of a novel HS 3-O-sulfotransferase isoform, designated as HS 3-O-sulfotransferase isoform 5 (3-OST-5). 3-OST-5 cDNA was isolated from a human placenta cDNA library and expressed in COS-7 cells. The disaccharide analysis of 3-OST-5-modified HS revealed that 3-OST-5 generated at least three 3-O-sulfated disaccharides as follows: IdoUA2S-AnMan3S, GlcUA-AnMan3S6S, and IdoUA2S-AnMan3S6S. Transfection of the plasmid expressing 3-OST-5 rendered wild type Chinese hamster ovary cells susceptible to the infection by herpes simplex virus 1, suggesting that 3-OST-5-modified HS serves as an entry receptor for herpes simplex virus 1. In addition, 3-OST-5-modified HS bound to herpes simplex viral envelope protein glycoprotein D. Furthermore, we found that 3-OST-5-modified HS also bound to antithrombin, suggesting that 3-OST-5 also produces anticoagulant HS. In summary, our results indicate that a new member of 3-OST family generates both anticoagulant HS and an entry receptor for herpes simplex virus 1. These results provide a new insight regarding the mechanism for the biosynthesis of biologically active HS.
Highlights
Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine residue of heparan sulfate (HS) to form 3-O-sulfated Heparan sulfates (HSs)
HS D-glucosaminyl 3-O-sulfotransferase (3-OST) isoforms generate 3-Osulfated glucosamine residues that are linked to different sulfated uronic acid residues. 3-OST-1 transfers sulfate to the 3-OH position of an N-sulfated glucosamine residue that is linked to a glucuronic acid residue at the nonreducing end type 1; gD, herpes envelope glycoprotein D; AT, antithrombin; HSact, anticoagulant-active or antithrombin-binding HS; HSinact, non-antithrombin-binding HS; 3-OST, HS D-glucosaminyl-3-O-sulfotransferase; IdoUA2S, L-iduronic acid 2-O-sulfate; AnMan, AnMan3S, AnMan6S, and AnMan3S6S, 2,5-anhydromannitol, 2,5-anhydromannitol 3-O-sulfate, 6-O-sulfate, and 3,6-O-disulfate; ConA, concanavalin A; CHO, Chinese hamster ovary; RPIP-HPLC, reverse-phase ion-pairing HPLC; MES, 2-(N-morphilino)ethanesulfonic acid; X-gal, 5-bromo-4-chloro3-indolyl--D-galactopyranoside
Isolation of the cDNA Encoding 3-O-sulfotransferase isoform 5 (3-OST-5)—Probing the nonredundant data base of National Center for Biotechnology Information with the deduced amino acid sequence of human 3-OST-1, we identified a 165-kb genomic clone with GenBankTM accession number AL355498
Summary
Recombinant human 3-OST-3A and mouse 3-OST-1 enzymes were expressed in Sf9 cells using a baculovirus expression system. Preparation of metabolically 35S-labeled HS from CHO cells was described elsewhere [23]. The 35S-labeled disaccharide standards, IdoUA2S-AnMan3S and IdoUA2S-AnMan3S6S, were prepared from low pH (pH 1.5) nitrous acid-degraded HS that was modified by purified 3-OST-3 enzyme as described by Liu et al [11]. By using Genscan, we predicted a 1,041-bp open reading frame that encodes a homologous protein from a genomic clone RP11-112L15 with GenBankTM accession number AL355498. The open reading frame sequence, designated as 3-OST-5, was located in two exons. Both 5Ј- and 3Ј-primers were synthesized based upon the sequence of exon 1 and exon 2, respectively. The isolated cDNA has an identical sequence that was predicted from the genomic clone. The isolated cDNA has an identical sequence that was predicted from the genomic clone. 5Ј-Rapid amplification of cDNA ends was performed but failed
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