Molecular cloning, expression and characterization of an Ascaris inhibitor for pepsin and cathepsin E.
Molecular cloning of a cDNA for a pepsin inhibitor in the round worm, Ascaris suum, was achieved. The ORF was found to encode a 20-residue potential signal peptide and a 149-residue inhibitor moiety. Northern analysis showed the mRNA for the inhibitor to be expressed in the body wall and not in the viscera. To obtain the active inhibitor, we constructed a yeast expression vector, pYES2API, containing the inducible galactosidase promoter and a DNA fragment encoding a fusion protein of the yeast alpha-factor leader and the Ascaris pepsin inhibitor. The active inhibitor was secreted in the culture medium, the yield being approximately 3 mg x l(-1) x day(-1), and purified by a two-step procedure that included HPLC. The inhibitor inactivated pepsin A and cathepsin E almost completely at amounts equimolar with the enzymes, but was 100-fold less effective against pepsin C and did not act on cathepsin D and renin. Ki values for the inhibition of pepsin A and cathepsin E were in the nanomolar range below pH 5. Since the inhibitor activity was lost by modification of specific Lys residues, including Lys110, an electrostatic interaction between these Lys residues and Asp/Glu residues of pepsin A or cathepsin E was thought to be essential for the inhibition.
Kageyama
T
T
Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Aichi, Japan. kageyama@smtp.pri.kyoto-u.ac.jp
D000595:Amino Acid Sequence; D000818:Animals; D017165:Ascaris suum / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020059:Cathepsin E; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010434:Pepsin A / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D002403:Cathepsins; D010434:Pepsin A; D020059:Cathepsin E