Molecular cloning and expression of a third member of the heparan sulfate/heparin GlcNAc N-deacetylase/N-sulfotransferase family

N-Deacetylation and N-sulfation of N-acetylglucosamine residues in heparan sulfate and heparin initiate a series of chemical modifications that ultima tely lead to oligosaccharide sequences with specific ligand binding propert ies. These reactions are catalyzed by GlcNAc N-deacetylase/N-sulfotransfera se (NDST), a monomeric enzyme with two catalytic activities. Two genes enco ding NDST isozymes have been described, one from rat liver (NDST1) and anot her from murine mastocytoma (NDST2), Both isozymes are expressed in tissues in varying amounts, but their relative contribution to heparan sulfate for mation in any one tissue is unknown. We now report the identification of a third member of the NDST family, designated NDST3, A full-length cDNA clone (3.2 kilobase pairs) encoding a 873-amino acid protein was obtained from a human fetal/infant brain cDNA library, Human NDST3 (hNDST3) has a nucleoti de sequence homologous but not identical to hNDST1 and NDST2. The deduced a mino acid sequence shows 70% and 65% amino acid identity to that of hNDST1 and NDST2, respectively. A soluble chimera of hNDST3 and protein A exhibite d both N-deacetylase and M-sulfotransferase activity, confirming its enzyma tic identity, Northern blot analysis of human fetal brain poly(A)(+) RNA sh owed a single transcript of 6.4 kilobase pairs, Reverse transcription polym erase chain reaction analysis revealed more restricted tissue expression of hNDST3 than hNDST1 and NDST2, and high levels in brain, liver, and kidney. Analysis of Chinese hamster ovary cells revealed expression of NDST1 and N DST2, but not NDST3. In a Chinese hamster ovary cell mutant exhibiting redu ced N-sulfotransferase activity and reduced sulfation of heparan sulfate (B ame, K. J., and Esko, J, D, (1989) J, Biol. Chem, 264, 8059-8065), expressi on of NDST1 was greatly reduced, but NDST2 was expressed normally, suggesti ng that both enzymes are involved in heparan sulfate assembly, The discover y of multiple NDST isozymes suggests that the assembly of heparan sulfate i s much complicated than previously appreciated.