Differences in specificity for the interactions of stefins A, B and D with cysteine proteinases

Abstract

Four different stefin-type cysteine proteinase inhibitors have been isolated from porcine thymus and skin. Amino acid sequence determination revealed the presence of stefin A and stefin B type inhibitors and two new inhibitors, designated as porcine stefin D1 and stefin D2. Stefin D1 was identified as PLCPI, an inhibitor recently characterized from porcine polymorphonuclear leukocytes [Lenarčič et al. (1993) FEBS Lett. 336, 289–292]. Stefin A is composed of 101 amino acids and has an M _r of 11391 while stefin B contains 98 amino acids, has an M _r of 11174 and is N-terminally blocked. All inhibitors were found to be fast-acting inhibitors of papain, cathepsin L and cathepsin S (K _i = 0.009–0.161 nM). Stefins A and B also bind tightly and rapidly to cathepsin H (K _i = 0.027 and 0.069 nM, respectively), while stefins D1 and D2 have been shown to be very poor inhibitors of cathepsin H (K _i = 102–150 nM). he decreased affinity of these inhibitors toward cathepsin B (K _i = 2–1700 nM) was shown to be mainly due to the low second order association rate constants. The presence of a highly negatively charged N-terminus on stefin D1 constitutes a likely structural determinant of inhibitor specificity.