MOLECULAR-CLONING AND EXPRESSION OF MOUSE AND HUMAN CDNAS ENCODING HEPARAN-SULFATE D-GLUCOSAMINYL 3-O-SULFOTRANSFERASE

The cellular rate of anticoagulant heparan sulfate proteoglycan (HSPG( act)) generation is determined by the level of a kinetically limiting microsomal activity, HSact conversion activity, which is predominantly composed of the long sought heparan sulfate D-glucosaminyl 3-O-sulfot ransferase (3-OST) (Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27063-27071; L iu, J., Shworak, N. W., Fritze, L. M. S., Edelberg, J. M., and Rosenbe rg, R. D. (1996) J. Biol. Chem. 271, 27072-27082). Mouse 3-OST cDNAs w ere isolated by proteolyzing the purified enzyme with Lys-C, sequencin g the resultant peptides as well as the existing amino terminus, emplo ying degenerate polymerase chain reaction primers corresponding to the sequences of the peptides as well as the amino terminus to amplify a fragment from LTA cDNA, and utilizing the resultant probe to obtain fu ll-length enzyme cDNAs from a lambda Zap Express LTA cDNA library. Hum an 3-OST cDNAs were isolated by searching the expressed sequence tag d ata bank with the mouse sequence, identifying a partial-length human c DNA and utilizing the clone as a probe to isolate a full-length enzyme cDNA from a lambda TriplEx human brain cDNA library. The expression o f wild-type mouse 3-OST as well as protein A-tagged mouse enzyme by tr ansient transfection of COS-7 cells and the expression of both wildtyp e mouse and human 3-OST by in vitro transcription/ translation demonst rate that the two cDNAs directly encode both HSact conversion and 3-OS T activities, The mouse 3-OST cDNAs exhibit three different size class es because of a 5'-untranslated region of variable length, which resul ts from the insertion of 0-1629 base pairs (bp) between residues 216 a nd 217; however, all cDNAs contain the same open reading frame of 933 bp. The length of the 3'-untranslated region ranges from 301 to 430 bp . The nucleic acid sequence of mouse and human 3-OST cDNAs are similar to 85% similar, encoding novel 311- and 307-amino acid proteins of 35 ,876 and 35,750 daltons, respectively, that are 93% similar. The encod ed enzymes are predicted to be intraluminal Golgi residents, pre-sumab ly interacting via their C-terminal regions with an integral membrane protein. Both 3-OST species exhibit five potential N-glycosylation sit es, which account for the apparent discrepancy between the molecular m asses of the encoded enzyme (similar to 34 kDa) and the previously pur ified enzyme (similar to 46 kDa). The two 3-OST species also exhibit s imilar to 50% similarity with all previously identified forms of the h eparan biosynthetic enzyme N-deacetylase/N-sulfotransferase, which sug gests that heparan biosynthetic enzymes share a common sulfotransferas e domain.