Biosynthesis of the prohormone convertase PC2 in Chinese hamster ovary cells and in rat insulinoma cells.
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.
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Seidah
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Lindberg
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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
D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D013381:Subtilisins; D045706:Proprotein Convertase 2