Expression of enzymatically active rat dipeptidyl peptidase IV in Chinese hamster ovary cells after transfection.
Dipeptidyl peptidase IV (DPPIV) is a cell surface membrane glycoprotein expressed in many tissues. We have subcloned the coding region of a full-length cDNA for DPPIV into the inducible eukaryotic expression vector pMSG. The resulting construct was used to transfect Chinese hamster ovary (CHO) cells. Stable transformants were found to express DPPIV, and the expression is enhanced by dexamethasone. Metabolic labeling of the transfected cells with [35S]Met followed by immunoprecipitation revealed the presence of two specific products of apparent Mr 100,000 (100-kDa form) and 110,000 (110-kDa form), respectively. Pulse-chase experiments demonstrated that the 100-kDa form can be chased into the 110-kDa form, suggesting the 100-kDa form is the precursor of the 110-kDa form. Further studies with endo H treatment demonstrated that the carbohydrate structures are of the high-mannose type, and of the complex type for the 100- and 110-kDa forms, respectively. The 110-kDa form is present at the cell surface as shown by its accessibility to cell surface iodination. The DPPIV expressed on the cell surface is resistant to digestion by relatively high concentrations of trypsin. Studies also demonstrated that the surface DPPIV is fairly stable with a half-life for turnover of about 40 h. Furthermore, the DPPIV produced in the transfected cells displays specific dipeptidyl peptidase activity. The stably transfected cells that express enzymatically active DPPIV in an inducible manner will provide an excellent system for further biochemical, functional, and cell biological characterizations of DPPIV.
Hong
W J
WJ
Department of Biological Sciences, State University of New York, Buffalo 14260.
Piazza
G A
GA
Hixson
D C
DC
Doyle
D
D
D000818:Animals; D001692:Biological Transport; D015152:Blotting, Northern; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D006224:Cricetinae; D003907:Dexamethasone / Q000494:pharmacology; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000096:biosynthesis* / Q000235:genetics; D004790:Enzyme Induction; D005260:Female; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015183:Restriction Mapping; D014162:Transfection; D014170:Transformation, Genetic; D014357:Trypsin / Q000378:metabolism
D012333:RNA, Messenger; D003907:Dexamethasone; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D014357:Trypsin