Multiple isoforms of heparan sulfate D-glucosaminyl 3-O-sulfotransferase -Isolation, characterization, and expression of human cDNAs and identification of distinct genomic loci

3-O-Sulfated glucosaminyl residues are rare constituents of heparan sulfate and are essential for the activity of anticoagulant heparan sulfate. Cellu lar production of the critical active structure is controlled by the rate-l imiting enzyme, heparan sulfate D-glucosaminyl 3-O-sulfotransferase-1 (3-OS T-1) (EC 2.8.2.23). We have probed the expressed sequence tag data base wit h the carboxyl-terminal sulfotransferase domain of 3-OST-1 to reveal three novel, incomplete human cDNAs. These were utilized in library screens to is olate full-length cDNAs. Clones corresponding to predominant transcripts we re obtained for the 367-, 406-, and 390-amino acid enzymes 3-OST-2, 3-OST-3 (A), and 3-OST-3(B), respectively. These type II integral membrane proteins are comprised of a divergent amino-terminal region and a very homologous c arboxyl-terminal sulfotransferase domain of similar to 260 residues. Also r ecovered were partial length clones for 3-OST-4. Expression of the full-len gth enzymes confirms the 3-O-sulfation of specific glucosaminyl residues wi thin heparan sulfate (Liu, J., Shworak, N. W., Sinay, P., Schwartz, J. J. Z hang, L., Fritze, L. M. S., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, 5185-5192). Southern analyses suggest the human 3OST1, 3OST2, and 3OST4 ge nes, and the corresponding mouse isologs, are single copy. However, 3OST3A and 3OST3B genes are each duplicated in humans and show at least one copy e ach in mice. Intriguingly, the entire sulfotransferase domain sequence of t he 3-OST-3(B) cDNA (774 base pairs) was 99.2% identical to the same region of 3-OST-3(A). Together, these data argue that the structure of this functi onally important region is actively maintained by gene conversion between 3 OST3A and 3OST3B loci. Interspecific mouse back-cross analysis identified t he loci for mouse 3Ost genes and syntenic assignments of corresponding huma n isologs were confirmed by the identification of mapped sequence-tagged si te markers, Northern blot analyses indicate brain exclusive and brain predo minant expression of 3-OST-4 and 3-OST-2 transcripts, respectively; whereas , 3-OST-3(A) and 3-OST-3(B) isoforms show widespread expression of multiple transcripts. The reiteration and conservation of the 3-OST sulfotransferas e domain suggest that this structure is a self-contained functional unit. M oreover, the extensive number of 3OST genes with diverse expression pattern s of multiple transcripts suggests that the novel 3-OST enzymes, like 3-OST -1, regulate important biologic properties of heparan sulfate proteoglycans .