QUAIL CYSTATIN - ISOLATION AND CHARACTERIZATION OF A NEW MEMBER OF THE CYSTATIN FAMILY AND ITS HYPOTHETICAL INTERACTION WITH CATHEPSIN-B

Quail cystatin, a new cysteine proteinase inhibitor protein of the cys tatin superfamily, was purified from egg albumen of Japanese quail Cot urnix coturnix japonica, Amino acid sequencing and mass spectrometry r evealed the complete 116 amino acid residue primary structure of a pho sphorylated form (13 173 Da). The inhibitor has a 90% sequence identit y with chicken cystatin, Its interaction with papain is rapid and tigh t (K-i = 4.4 pM; k(on) = 1.8 x 10(7) M-1 s(-1); k(off) = 0.8 x 10(-4) s(-1)) and very similar to that of chicken cystatin, Surprisingly, how ever, cathepsin B was inhibited 15-fold more strongly by quail cystati n (K-i = 47 pM; k(on) = 19 x 10(7) M-1 s(-1); k(off) = 9 x 10(-4) s(-1 )) than by chicken cystatin (K-i = 784 pM; k(on) = 2.9 x 10(7) M-1 s(- 1); k(off) = 24 x 10(-4) s(-1)), Intuitive comparative conformational inspection of related inhibitors and of cognate enzymes suggest that: (i) the 3D structure of quail cystatin is nearly identical to that of chicken cystatin, (ii) quail cystatin can interact with cathepsin B an alogous to the stefin B-papain interaction, if the 'occluding loop' of cathepsin B possesses an 'open' conformation, (iii) the greater inhib ition of cathepsin B by quail cystatin compared to chicken cystatins p robably arises from two additional ionic interactions between residues Arg(15) and Lys(112) of the inhibitor and Glu(194) and Asp(124) of th e enzyme, respectively. The two potential salt bridges are located out side of the known contact regions between cystatins and peptidases of the papain family. (C) 1997 Federation of European Biochemical Societi es.