Production and characterization of two variants of human cystatin SA encoded by two alleles at the CST2 locus of the type 2 cystatin gene family.
Two variants of cystatin SA encoded by two alleles at the CST2 locus of the type 2 human cystatin gene family were expressed in Escherichia coli. One, termed cystatin SA1, is identical to cystatin SA [S. Isemura, E. Saitoh, and K. Sanada J. Biochem. 102, 693-704, 1987]. Another, termed cystatin SA2, carries two amino acid substitutions (59Gly-->Asp; 120Glu-->Asp), one of which is in the so-called QXVXG region (the first hairpin loop) and another in the C-terminal portion of the molecule. Four recombinant cystatins [full-sized cystatin SA1, two N-terminally truncated cystatin SA1 lacking four residues (WSPQ) and six residues (WSPQEE), and full-sized cystatin SA2] were purified from the periplasmic fractions of E. coli cells. Two N-terminally truncated recombinant cystatin SA1 inhibited bovine cathepsin C with 2- to 20-fold lower Ki values than that of the full-sized one. In the inhibition of papain and ficin, however, both of the N-terminally truncated cystatin SA1 displayed a 10-fold higher Ki value than that of full-sized one. In the inhibition of papain, ficin, and recombinant human cathepsin K, recombinant cystatin SA2 showed, respectively, 3826-, 1090-, and 30-fold higher Ki values compared with those of SA1. Recombinant cystatin SA2 inhibited bovine cathepsin C with a 50-fold lower Ki value compared with that of SA1. Recombinant cystatin SA1 did not inhibit human cathepsin H but SA2 inhibited it slightly (Ki = 528 nM). Neither of the recombinant variants inhibited bovine cathepsin B. Our data supply evidence indicating that the amino acid sequence of the first hairpin loop of the cystatin superfamily is important in the inhibition of papain, ficin, cathepsin C, cathepsin H, and cathepsin K.
Saitoh
E
E
Department of Oral Biochemistry, Nippon Dental University, School of Dentistry at Niigata, Japan.
Minaguchi
K
K
Ishibashi
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D002417:Cattle; D015891:Cystatins / Q000737:chemistry*; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005361:Ficain / Q000037:antagonists & inhibitors; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors; D010446:Peptide Fragments / Q000494:pharmacology; D011480:Protease Inhibitors / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012463:Saliva / Q000737:chemistry; D055333:Salivary Cystatins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
C525172:CST2 protein, human; D015891:Cystatins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D055333:Salivary Cystatins; D002403:Cathepsins; D010450:Endopeptidases; D010206:Papain; D005361:Ficain