title: Biosynthesis of the prohormone convertase PC2 in Chinese hamster ovary cells and in rat insulinoma cells.
abstract: The biosynthesis of the prohormone convertase PC2 was studied in Chinese hamster ovary cells stably transfected with PC2 cDNA (CHO/PC2) and in rat insulinoma cells (Rin5f). The major form of PC2 synthesized by CHO/PC2 cells was a 75-kDa protein corresponding to proPC2; this protein was retained intracellularly for 2-4 h following synthesis, suggesting prolonged intracellular residence. In contrast, the major form of PC2 within Rin cells initially exhibited a molecular mass of 72 kDa and was then progressively converted to a 64-kDa species. This 64-kDa species, which required 1-2 h to be released, was the major PC2 form detectable in Rin cell medium. Calcium-dependent benzyloxycarbonyl-Arg-Ser-Lys-Arg-aminomethylcoumarin cleaving activity was found in spent Rin cell medium; this activity could be immunoprecipitated with a carboxyl-terminal PC2 antibody, but not with preimmune serum. In neither cell line did intracellular PC2 become endoglycosidase H-resistant over time. PC2 released from Rin cells was also endoglycosidase H-sensitive. Microsequencing and endoglycosidase H results indicate that 75-kDa CHO cell PC2 and 72-kDa Rin cell PC2 both represent proPC2. We speculate that (a) PC2 undergoes unusual glycosylation, which may be related to its slow release from cells, and (b) the 64-kDa molecule detectable in spent Rin cell medium represents the enzymatically active form of PC2.
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
authors.forename: F S
authors.initials: FS
authors.lastname: Shen
authors.forename: N G
authors.initials: NG
authors.lastname: Seidah
authors.forename: I
authors.initials: I
authors.lastname: Lindberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D016521:Electrophoresis, Gel, Pulsed-Field; D007340:Insulinoma; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D045706:Proprotein Convertase 2; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D013381:Subtilisins / Q000096:biosynthesis* / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D013381:Subtilisins; D045706:Proprotein Convertase 2