Site-directed mutagenesis of human prorenin. Substitution of three arginine residues in the propeptide with glutamine residues yields active prorenin.
Human prorenin is an inactive zymogen comprising 43 amino acid residues at the amino terminus of human renin. The aim of this work was to determine why prorenin is inactive at neutral pH. Eighteen different mutant prorenins, in which positively charged residues in the propeptide were substituted with either glutamine (Gln) or lysine (Lys) residues by site-directed mutagenesis, were expressed in COS-7 cells and characterized. By replacing each of the three arginine (Arg) residues (Arg10P, Arg15P, and Arg20P) with Gln residues, partially active prorenins were produced, which exhibited significant but not full renin activity without trypsin activation. The effect of double or triple amino acid substitutions on the appearance of active prorenin was cumulative, the activity reaching about 80% in a mutant in which all the three Arg residues were replaced by Gln residues. In contrast, mutant prorenins with Lys residues substituted for the Arg residues were inactive. These results clearly indicate that the positive charges of the three Arg residues are essential for maintenance of the human prorenin in an inactive form.
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki.
T
T
Yamauchi
M
M
Nagahama
T
T
Watanabe
Y
Y
Ishizuka
H
H
Hori
K
K
Murakami
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D016282:Aspartic Acid Endopeptidases; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D010450:Endopeptidases; D004792:Enzyme Precursors / Q000235:genetics* / Q000378:metabolism; D005973:Glutamine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D015202:Protein Engineering; D051381:Rats; D012083:Renin / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013552:Swine
D004792:Enzyme Precursors; D005973:Glutamine; D001120:Arginine; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; D012083:Renin