Heparin has a higher content of N-sulfated glucosamine and L-iduronic acid than heparan sulfate. Deacetylation of N-acetylglucosamine followed by N-sulfation may be important steps differentiating the biosynthesis of these glycosaminoglycans. We have cloned, by cross-hybridization with the cDNA from rat liver heparan sulfate N-deacetylase/N-sulfotransferase, a protein from a heparin synthesizing mastocytoma derived cell line called MST. This protein, which has both N-deacetylase/N-sulfotransferase activities, has a predicted amino acid sequence homology of 70% with the above rat liver enzyme and is unique for the following reasons. 1) It was found to be encoded by a 3.8-kilobase mRNA that was unique to heparin-producing cells; an 8.5-kilobase mRNA encoding the rat liver enzymes has been found to occur in all mammalian cells tested on the basis of nucleic acid cross-hybridization; 2) the protein overexpressed in COS cells in its full-length transmembrane form or as a soluble secreted protein A chimera displayed ratios of N-deacetylase to N-sulfotransferase activities that were 4-8-fold higher than that observed for the enzyme found in liver that is involved in the biosynthesis of heparan sulfate. These results suggest that the MST-derived enzyme is probably unique to the production of heparin in mast cells.

N-Heparan sulfate sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the nitrogen of glucosamine in heparan sulfate. The enzyme has been previously purified to apparent homogeneity from rat liver (Brandan, E., and Hirschberg, C. B. (1988) J. Biol. Chem. 263, 2417-2422). We have now cloned the rat liver enzyme using the following strategy: (a) the amino acid sequence was obtained from tryptic peptides of the purified protein, (b) mixed oligonucleotides were generated based on the sequence of the tryptic peptides, (c) a polymerase chain reaction fragment was obtained using mixed oligonucleotide interprimer amplification of cDNA, and (d) this fragment was used to screen rat liver lambda gt 10 and lambda ZAP libraries. Three clones were obtained, one of which seems to contain the complete coding sequence of the N-heparan sulfate sulfotransferase (N-HSST). Evidence that the cDNA clone corresponds to the previously purified and characterized N-HSST was the following: (a) the predicted sequence of the N-HSST contains all of the 11 tryptic peptides obtained from the purified protein, (b) when a cDNA containing the sequence coding for the N-HSST was introduced in a eukaryotic expression vector and transfected in COS-1 cells, the enzyme activity was expressed 9-fold over controls, and (c) the characteristic of the predicted protein fits with the purified protein in terms of molecular weight, membrane localization, and its being an N-linked glycoprotein. The size of the longest cDNA isolated is 4.1 kilobases, which is in close agreement with the 4.2-kilobase size of one of the mRNA observed in Northern analyses. In addition, messages of 7.0 and 8.5 kilobases were also observed, suggesting that a large portion is untranslated. The latter messages were the major mRNA species detected.