title: 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.
abstract: Chicken cystatin and human kininogen domain 2 are members of the cystatin superfamily of protein-type cysteine proteinase inhibitors. They show structural and functional similarities, but only human kininogen domain 2 inhibits calpain. Using recombinant chicken cystatin as a scaffold 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 single and combined manner. Seven hybrids were expressed, purified to homogeneity, characterized protein-chemically, and their inhibition of papain, actinidin, human cathepsin B, human cathepsin L and calpain (80-kDa subunit of rabbit skeletal muscle calpain II and porcine erthrocyte calpain 1) was determined. Strong but temporary inhibition of calpain by chicken cystatin hybrids carrying the N-terminus alone (variant sc1-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 inhibitors. These data indicate that the inhibiton of calpain by human kininogen domain 2 requires the correct conformation and combination of several contact sites, and suggest that the N-terminus and the first hairpin 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 with the impaired inhibition of cathepsin L, papain, actinidin, cathepsin B and calpain by the hybrids sc1/3-KD2, sc2/3-KD2 and sc1/2/3-KD2, these results support our hypothesis that all three predicted contact regions of kininogen domain 2 contribute to binding in the active-site clefts of papain-like enzymes in a finely balanced manner.
authors.affiliation: Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik and Poliklinik, Klinikum Innenstadt der LMU München, Germany.
authors.forename: E A
authors.initials: EA
authors.lastname: Auerswald
authors.forename: D K
authors.initials: DK
authors.lastname: Nägler
authors.forename: S
authors.initials: S
authors.lastname: Gross
authors.forename: I
authors.initials: I
authors.lastname: Assfalg-Machleidt
authors.forename: M T
authors.initials: MT
authors.lastname: Stubbs
authors.forename: C
authors.initials: C
authors.lastname: Eckerskorn
authors.forename: W
authors.initials: W
authors.lastname: Machleidt
authors.forename: H
authors.initials: H
authors.lastname: Fritz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002154:Calpain / Q000037:antagonists & inhibitors; D002401:Cathepsin B / Q000037:antagonists & inhibitors; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors; D002645:Chickens; D015891:Cystatins / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000187:drug effects; D010450:Endopeptidases; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007704:Kininogens / Q000737:chemistry*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010206:Papain / Q000037:antagonists & inhibitors; D017386:Sequence Homology, Amino Acid
chemical_list: D015891:Cystatins; D004791:Enzyme Inhibitors; D007704:Kininogens; D009838:Oligodeoxyribonucleotides; D002403:Cathepsins; D010450:Endopeptidases; D002154:Calpain; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C018745:actinidain; C534281:CTSL protein, human; D056668:Cathepsin L; D010206:Papain