Proinsulin endopeptidase substrate specificities defined by site-directed mutagenesis of proinsulin.
Two endopeptidases are involved in the conversion of proinsulin; a type I activity directed at the B chain, Arg31,Arg32, C-peptide junction, and type II which cleaves the C-peptide, Lys64,Arg65, A chain junction. To define further the substrate specificities of these enzymes, a series of mutant preproinsulin cDNAs were generated by site-directed and deletion mutagenesis. These were inserted into pT7 plasmids and capped cRNA transcripts synthesized, that were then microinjected into Xenopus oocytes. Oocytes were biosynthetically radiolabeled with [3H]leucine and the secreted peptides (greater than 95% present as unprocessed proinsulins) then incubated with types I and II endopeptidase activities prepared from isolated insulinoma secretory granules. The reaction products were analyzed by high performance liquid chromatography. Des-38-62-proinsulin, in which all but six amino acids of C-peptide were deleted was not processed by either enzyme. The mutant Lys64,Arg65 to Thr64,Arg65 was not cleaved by the type II enzyme but was still a substrate for the type I enzyme. The mutant Arg31,Arg32 to Arg31,Gly32 correspondingly was not cleaved by the type I enzyme; however, in this case it was not attacked by the type II enzyme. These results indicate that not only is the presence of a dibasic sequence essential, but also that the secondary structure of the protein is important in determining whether the prohormone is susceptible to proteolytic processing.
Docherty
K
K
Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, United Kingdom.
Rhodes
C J
CJ
Taylor
N A
NA
Shennan
K I
KI
Hutton
J C
JC
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism*; D015870:Gene Expression; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics; D018075:RNA, Complementary; D013379:Substrate Specificity; D014162:Transfection; D014981:Xenopus
D018075:RNA, Complementary; D012313:RNA; D004247:DNA; D011384:Proinsulin; D010450:Endopeptidases; C054196:proinsulin endopeptidase I; C061496:proinsulin endopeptidase II