Chicken egg white cystatin was first described in the late 1960s. Since then, our knowledge about a superfamily of similar proteins present in mammals, birds, fish, insects, plants and some protozoa has expanded, and their properties as potent peptidase inhibitors have been firmly established. Today, 12 functional chicken cystatin relatives are known in humans, but a few evolutionarily related gene products still remain to be characterized. The type 1 cystatins (A and B) are mainly intracellular, the type 2 cystatins (C, D, E/M, F, G, S, SN and SA) are extracellular, and the type 3 cystatins (L- and H-kininogens) are intravascular proteins. All true cystatins inhibit cysteine peptidases of the papain (C1) family, and some also inhibit legumain (C13) family enzymes. These peptidases play key roles in physiological processes, such as intracellular protein degradation (cathepsins B, H and L), are pivotal in the remodelling of bone (cathepsin K), and may be important in the control of antigen presentation (cathepsin S, mammalian legumain). Moreover, the activities of such peptidases are increased in pathophysiological conditions, such as cancer metastasis and inflammation. Additionally, such peptidases are essential for several pathogenic parasites and bacteria. Thus cystatins not only have capacity to regulate normal body processes and perhaps cause disease when down-regulated, but may also participate in the defence against microbial infections. In this chapter, we have aimed to summarize our present knowledge about the human cystatins.

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 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 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 K i values 0.2-0.3 nM. Complexes between cystatin C and papain inhibited legumain activity against benzoyl-AsnNHPhNO 2 as efficiently as did cystatin C alone. Conversely, cystatin C inhibited papain activity against benzoyl-Arg-NHPhNO 2 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 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 molecular modeling led to the suggestion that a loop located on the opposite side to the papain-binding surface, between the ␣-helix and the first strand of the main ␤-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 Asn 39 residue in this loop (N39K-cystatin C); this variant showed a slight reduction in affinity for cathepsin B (K i 1.5 nM) but > >5,000-fold lower affinity for legumain (K i > >1,000 nM) than wild-type cystatin C.

The residues of the inhibitor are labeled without a chain designator. The residues of the enzyme are marked "e", except for the first residue, marked "p" (E1p), which belongs to the prosegment sequence.Abbreviations used: E-64, L-trans-epoxysuccinylleucylamido-(4-guanidino)butane; MAD, multiwavelength anomalous diffraction; MS, mass spectrometry; PDB, Protein Data Bank; NHMec, 7-(4-methyl)coumarylamide; r.m.s., root mean square; Z-, carboxybenzoyl-; GST, glutathione-S-transferaseChagasin-cathepsin L complex structure 3 SUMMARY

Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared with its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg 26 -cystatin D and solved its structures at room temperature and at cryo conditions to 2.5-and 1.8-Å resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded antiparallel ␤-sheet wrapped around a five-turn ␣-helix. The structures reveal differences in the peptidase-interacting regions when compared with other cystatins, providing plausible explanations for the restricted inhibitory specificity of cystatin D for some papain-like peptidases and its lack of reactivity toward legumainrelated enzymes.

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