Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site

We have investigated the inhibition of the recently identified family C13 c ysteine peptidase, pig legumain, by human cystatin C, The cystatin was seen to inhibit enzyme activity by stoichiometric 1:1 binding in competition wi th substrate. The K-i 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 C 1 cysteine peptidase, cathepsin B, However, cystatin C variants with altera tions in the N-terminal region and the "second hairpin loop" that rendered the cystatin inactive against cathepsin B, still inhibited legumain with K- i values 0.2-0.3 nM. Complexes between cystatin C and papain inhibited legu main activity against benzoyl-Asn-NHPhNO2, as efficiently as did cystatin C alone, Conversely, cystatin C inhibited papain activity against benzoyl-Ar g-NHPhNO2 whether or not the cystatin had been incubated with legumain, str ongly indicating that the cystatin inhibited the two enzymes with non-overl apping sites. A ternary complex between legumain, cystatin C, and papain wa s demonstrated by gel filtration supported by immunoblotting. Screening of a panel of cystatin superfamily members showed that type 1 inhibitors (cyst atins A and B) and low M-r 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 (K-i 0.0016 nM) and relatively weak (K -i 10 nM) affinity for legumain, respectively. Sequence alignments and mole cular modeling led to the suggestion that a loop located on the opposite si de to the papain-binding surface, between the alpha-helix and the first str and of the main beta-pleated sheet of the cystatin structure, could be invo lved in legumain binding. This was corroborated by analysis of a cystatin C variant with substitution of the Asn(39) residue in this loop (N39K-cystat in C); this variant showed a slight reduction in affinity for cathepsin B ( K-i 1.5 nM) but much greater than 5,000-fold lower affinity for legumain (K -i much greater than 1,000 nM) than wild-type cystatin C.