HYBRIDS OF CHICKEN CYSTATIN WITH HUMAN KININOGEN DOMAIN 2 SEQUENCES EXHIBIT NOVEL INHIBITION OF CALPAIN, IMPROVED INHIBITION OF ACTINIDIN AND IMPAIRED INHIBITION OF PAPAIN, CATHEPSIN-L AND CATHEPSIN-B

Chicken cystatin and human kininogen domain 2 are members of the cysta tin superfamily of protein-type cysteine proteinase inhibitors. They s how structural and functional similarities, but only human kininogen d omain 2 inhibits calpain. Using recombinant chicken cystatin as a scaf fold for hybrid cassette analysis, the known reactive-site regions (N- terminus, first hairpin loop and second hairpin loop) were substituted by the corresponding sequences of human kininogen domain 2 in a singl e and combined manner, Seven hybrids were expressed, purified to homog eneity, characterized protein-chemically, and their inhibition of papa in, actinidin, human cathepsin B, human cathepsin L and calpain (80-kD a subunit of rabbit skeletal muscle calpain II and porcine erythrocyte calpain I) was determined, Strong but temporary inhibition of calpain by chicken cystatin hybrids carrying the N-terminus alone (variant sc 1-KD2) or the N-terminus together with the first hairpin loop (variant sc1/2-KD2) was observed; hybrids of the second hairpin loop (sc3-KD2. sc1/3-KD2, sc2/3-KD2, sc1/2/3-KD2) were less strong calpain inhibitor s. These data indicate that the inhibition of calpain by human kininog en domain 2 requires the correct conformation and combination of sever al contact sites, and suggest that the N-terminus and the first hairpi n loop play a major role in this ensemble. Remarkably, hybrid sc2-KD2 exhibited 5 or 150 times stronger inhibition of actinidin compared to native chicken cystatin or to proteolytically isolated human kininogen domain 2, respectively. This indicates an important role of the first hairpin loop of cystatins in the interaction with actinidin. Along wi th the impaired inhibition of cathepsin L, papain, actinidin, cathepsi n B and calpain by the hybrids sc1/3-KD2, sc2/3-KD2 and sc1/2/3-KD2, t hese results support our hypothesis that all three predicted contact r egions of kininogen domain 2 contribute to binding in the active-site clefts of papain-like enzymes in a finely balanced manner.