title: Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site.
abstract: We have investigated the inhibition of the recently identified family C13 cysteine peptidase, pig legumain, by human cystatin C. The cystatin was seen to inhibit enzyme activity by stoichiometric 1:1 binding in competition with substrate. The Ki value for the interaction was 0.20 nM, i.e. cystatin C had an affinity for legumain similar to that for the papain-like family C1 cysteine peptidase, cathepsin B. However, cystatin C variants with alterations in the N-terminal region and the "second hairpin loop" that rendered the cystatin inactive against cathepsin B, still inhibited legumain with Ki values 0.2-0.3 nM. Complexes between cystatin C and papain inhibited legumain activity against benzoyl-Asn-NHPhNO2 as efficiently as did cystatin C alone. Conversely, cystatin C inhibited papain activity against benzoyl-Arg-NHPhNO2 whether or not the cystatin had been incubated with legumain, strongly indicating that the cystatin inhibited the two enzymes with non-overlapping sites. A ternary complex between legumain, cystatin C, and papain was demonstrated by gel filtration supported by immunoblotting. Screening of a panel of cystatin superfamily members showed that type 1 inhibitors (cystatins A and B) and low Mr kininogen (type 3) did not inhibit pig legumain. Of human type 2 cystatins, cystatin D was non-inhibitory, whereas cystatin E/M and cystatin F displayed strong (Ki 0.0016 nM) and relatively weak (Ki 10 nM) affinity for legumain, respectively. Sequence alignments and molecular modeling led to the suggestion that a loop located on the opposite side to the papain-binding surface, between the alpha-helix and the first strand of the main beta-pleated sheet of the cystatin structure, could be involved in legumain binding. This was corroborated by analysis of a cystatin C variant with substitution of the Asn39 residue in this loop (N39K-cystatin C); this variant showed a slight reduction in affinity for cathepsin B (Ki 1.5 nM) but >>5,000-fold lower affinity for legumain (Ki >>1,000 nM) than wild-type cystatin C.
authors.affiliation: Department of Clinical Chemistry, Institute of Laboratory Medicine, Lund University Hospital, S-221 85 Lund, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Alvarez-Fernandez
authors.forename: A J
authors.initials: AJ
authors.lastname: Barrett
authors.forename: B
authors.initials: B
authors.lastname: Gerhartz
authors.forename: P M
authors.initials: PM
authors.lastname: Dando
authors.forename: J
authors.initials: J
authors.lastname: Ni
authors.forename: M
authors.initials: M
authors.lastname: Abrahamson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D055316:Cystatin C; D015891:Cystatins / Q000378:metabolism / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010206:Papain / Q000378:metabolism; D010940:Plant Proteins; D016415:Sequence Alignment; D013552:Swine; D000099240:Asparaginyl Endopeptidase
chemical_list: D055316:Cystatin C; D015891:Cystatins; D003546:Cysteine Endopeptidases; D010206:Papain; D010940:Plant Proteins; D000099240:Asparaginyl Endopeptidase; C525030:CST3 protein, human