Molecular cloning and expression of a glycosaminoglycan N-acetylglucosaminyl N-deacetylase/N-sulfotransferase from a heparin-producing cell line.
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.
Orellana
A
A
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01655.
Hirschberg
C B
CB
Wei
Z
Z
Swiedler
S J
SJ
Ishihara
M
M
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D006493:Heparin / Q000096:biosynthesis*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D012515:Mast-Cell Sarcoma; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015238:Sulfotransferases / Q000096:biosynthesis* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D012333:RNA, Messenger; D006493:Heparin; D015238:Sulfotransferases; C021350:heparitin sulfotransferase; D000581:Amidohydrolases