Inhibition of Mammalian Legumain by Some Cystatins Is Due to a Novel Second Reactive Site
1999; Elsevier BV; Volume: 274; Issue: 27 Linguagem: Inglês
10.1074/jbc.274.27.19195
ISSN1083-351X
AutoresMarcia Alvarez-Fernandez, A. John Barrett, Bernd Gerhartz, Pam M. Dando, Jian Ni, Magnus Abrahamson,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoWe 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. TheK 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-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 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 α-helix and the first strand of the main β-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 (K i 1.5 nm) but ≫5,000-fold lower affinity for legumain (K i ≫1,000 nm) than wild-type cystatin C. 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. TheK 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-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 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 α-helix and the first strand of the main β-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 (K i 1.5 nm) but ≫5,000-fold lower affinity for legumain (K i ≫1,000 nm) than wild-type cystatin C. low molecular mass kininogen carboxymethyl- high pressure liquid chromatography 7-(4-methyl)coumarylamide polyacrylamide gel electrophoresis size exclusion chromatography poly(vinylidene) difluoride trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane polymerase chain reaction 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid benzoyl benzyloxycarbonyl The activities of cysteine peptidases of the papain family (C1) such as cathepsins B, H, L, S, and K in and around mammalian cells are regulated by reversible, tight-binding protein inhibitors of the cystatin superfamily (1Abrahamson M. Methods Enzymol. 1994; 244: 685-700Crossref PubMed Scopus (182) Google Scholar). The cystatins constitute a superfamily of evolutionarily related proteins that are all composed of at least one 100–120-residue domain with conserved sequence motifs (2Rawlings N.D. Barrett A.J. J. Mol. Evol. 1990; 30: 60-71Crossref PubMed Scopus (267) Google Scholar). The single-domain human members of this superfamily are of two major types. The type 1 cystatins (or stefins) A and B contain approximately 100 amino acid residues, lack disulfide bridges, and are synthesized without signal peptides. Cystatins of type 2 are secreted proteins of approximately 120 amino acid residues (M r13,000–14,000) and contain at least two characteristic intrachain disulfide bonds. The type 2 cystatins include the human cystatins C, D, S, SN, and SA, which are all products of genes located in the cystatin multigene locus on chromosome 20 (3Schnittger S. Rao V.V. Abrahamson M. Hansmann I. Genomics. 1993; 16: 50-55Crossref PubMed Scopus (64) Google Scholar). Two recently identified type 2 cystatins, cystatin E/M and cystatin F (also called leukocystatin), are also secreted low M r proteins but are more atypical in that they are glycoproteins and show only 30–35% sequence identity in alignments with the classical type 2 cystatins. They are, however, still functional inhibitors of family C1 cysteine peptidases (4Ni J. Abrahamson M. Zhang M. Fernandez M.A. Grubb A. Su J. Yu G.-L. Li Y. Parmelee D. Xing L. Coleman T.A. Gentz S. Thotakura R. Nguyen N. Hesselberg M. Gentz R. J. Biol. Chem. 1997; 272: 10853-10858Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 5Ni J. Fernandez M.A. Danielsson L. Chillakuru R.A. Zhang J. Grubb A. Su J. Gentz R. Abrahamson M. J. Biol. Chem. 1998; 273: 24797-24804Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 6Sotiropoulou G. Anisowicz A. Sager R. J. Biol. Chem. 1997; 272: 903-910Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 7Halfon S. Ford J. Foster J. Dowling L. Lucian L. Sterling M. Xu Y. Weiss M. Ikeda M. Liggett D. Helms A. Caux C. Lebecque S. Hannum C. Menon S. McClanahan T. Gorman D. Zurawski G. J. Biol. Chem. 1998; 273: 16400-16408Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). It has been shown that the cystatin inhibition of cysteine peptidases of the papain family is due to a tripartite wedge-shaped structure with very good complementarity to the active site clefts of such enzymes (8Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar). The three parts of the cystatin polypeptide chain included in the enzyme-binding domain are the N-terminal segment, a central loop-forming segment with the motif Gln-Xaa-Val-Xaa-Gly, and a second C-terminal hairpin loop typically containing a Pro-Trp pair (8Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar, 9Abrahamson M. Ritonja A. Brown M.A. Grubb A. Machleidt W. Barrett A.J. J. Biol. Chem. 1987; 262: 9688-9694Abstract Full Text PDF PubMed Google Scholar, 10Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar).Legumain (EC 3.4.22.34) is a cysteine endopeptidase that was until recently known only from plants (11Kembhavi A.A. Buttle D.J. Knight C.G. Barrett A.J. Arch. Biochem. Biophys. 1993; 303: 208-213Crossref PubMed Scopus (158) Google Scholar, 12Hara-Nishimura I. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 746-749Google Scholar) and Schistosoma(13Dalton J.P. Brindley P.J. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 749-754Google Scholar). In plants there is abundant evidence that legumain performs a protein-processing function, causing limited proteolysis of precursor proteins and protein splicing (12Hara-Nishimura I. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 746-749Google Scholar, 14Min W. Jones H. Struct. Biology. 1994; 1: 502-504Crossref Scopus (58) Google Scholar). Following the discovery of the enzyme in mammalian cells, it was cloned and sequenced from human (15Chen J.-M. Dando P.M. Rawlings N.D. Brown M.A. Young N.E. Stevens R.A. Hewitt E. Watts C. Barrett A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar) and mouse (16Chen J.M. Dando P.M. Stevens R.A.E. Fortunato M. Barrett A.J. Biochem. J. 1998; 335: 111-117Crossref PubMed Scopus (112) Google Scholar). The amino acid sequences of legumains show that they belong to a distinct family of cysteine endopeptidases (C13). Mammalian legumain is predominantly lysosomal in distribution (16Chen J.M. Dando P.M. Stevens R.A.E. Fortunato M. Barrett A.J. Biochem. J. 1998; 335: 111-117Crossref PubMed Scopus (112) Google Scholar), but its strict specificity for the hydrolysis of bonds on the carboxyl side of asparagine is very different from that of any cathepsin and adapts it particularly for limited proteolysis (17Dando P.M. Fortunato M. Smith L. Knight C.G. McKendrick J.E. Barrett A.J. Biochem. J. 1999; 339: 743-749Crossref PubMed Scopus (61) Google Scholar). Human legumain may have an important physiological function as a key enzyme in antigen presentation (18Manoury B. Hewitt E.C. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (303) Google Scholar).It was recently reported that pig legumain is inhibited by human cystatin C and chicken cystatin with K i values below 5 nm (15Chen J.-M. Dando P.M. Rawlings N.D. Brown M.A. Young N.E. Stevens R.A. Hewitt E. Watts C. Barrett A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). This finding was unexpected, since the cystatins are already known as potent inhibitors of the papain-like cysteine peptidases in the unrelated family C1. The legumain family members are believed to have a protein fold quite unlike that of papain, and to be much more closely related to the caspases and gingipain (19Chen J.-M. Rawlings N.D. Stevens R.A.W. Barrett A.J. FEBS Lett. 1998; 441: 361-365Crossref PubMed Scopus (194) Google Scholar). Although the active site cysteine residue could seem to be a common factor, it is known not to be required for the interaction of papain with cystatins (20Björk I. Ylinenjarvi K. Biochem. J. 1989; 260: 61-68Crossref PubMed Scopus (37) Google Scholar). The present investigation was undertaken to elucidate the mechanism of inhibition of mammalian legumain by cystatins.DISCUSSIONThe aim of the present investigation was to study the mechanism of inhibition of mammalian legumain by cystatins, to clarify how the inhibitor structure can result in tight-binding inhibition of enzymes belonging to two entirely different enzyme families, namely the papain family (C1) and the legumain family (C13). The different arrangements of catalytic residues and different active site motifs show that the two families are evolutionarily unrelated, and that their peptidases have different protein folds (19Chen J.-M. Rawlings N.D. Stevens R.A.W. Barrett A.J. FEBS Lett. 1998; 441: 361-365Crossref PubMed Scopus (194) Google Scholar). Moreover, legumain is not inhibited by the general inhibitor of enzymes belonging to family C1, E-64 (15Chen J.-M. Dando P.M. Rawlings N.D. Brown M.A. Young N.E. Stevens R.A. Hewitt E. Watts C. Barrett A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar), which supports the theory that the general topography of the active site clefts of legumains are probably entirely different from those of family C1 enzymes. Our present SEC, electrophoretic and enzyme kinetic results show that cystatin C can inhibit mammalian legumain, as cystatins inhibit family C1 enzymes (52Nicklin M.J.H. Barrett A.J. Biochem. J. 1984; 223: 245-253Crossref PubMed Scopus (141) Google Scholar), by high affinity reversible binding (K i 0.20 nm), in a bimolecular reaction that is competitive with substrate, and with no detectable cleavage of the cystatin in the legumain complex. Despite these similarities, our present results demonstrate that the mechanisms of inhibition of legumain and family C1 endopeptidases must be completely different.From structural studies of several cystatins (8Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar, 9Abrahamson M. Ritonja A. Brown M.A. Grubb A. Machleidt W. Barrett A.J. J. Biol. Chem. 1987; 262: 9688-9694Abstract Full Text PDF PubMed Google Scholar, 10Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar), it is well known that the N-terminal segment together with the "first and second hairpin loops" in cystatins are responsible for the inhibition of the C1 enzymes (Fig. 5). Consequently, removal of the N-terminal segment or substitution of any of the conserved amino acids in the N-terminal segment or the hairpin loops by Gly/Ala residues abolishes or seriously affects the inhibition of papain-like enzymes (26Hall A. Håkansson K. Mason R.W. Grubb A. Abrahamson M. J. Biol. Chem. 1995; 270: 5115-5121Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 53Auerswald E.A. Genenger G. Assfalg-Machleidt I. Machleidt W. Engh R.A. Fritz H. Eur. J. Biochem. 1992; 209: 837-845Crossref PubMed Scopus (50) Google Scholar). Of four such variants analyzed in the present study, all displayed virtually unaltered binding of legumain. Dimeric cystatin C, which is completely inactive against papain-like enzymes and by NMR studies has been shown to be a result of intermolecular interactions between the papain-binding surfaces of two cystatin C molecules (47Ekiel I. Abrahamson M. Fulton D.B. Lindahl P. Storer A.C. Levadoux W. Lafrance M. Labelle S. Pomerleau Y. Groleau D. LeSauteur L. Gehring K. J. Mol. Biol. 1997; 271: 266-277Crossref PubMed Scopus (99) Google Scholar), still showed legumain inhibition. We believe that this, together with the enzyme kinetic results presented and the direct demonstration of a ternary complex by SEC, proves that the binding sites for papain and legumain on cystatin C likely are completely independent of each other.Where then is the legumain reactive site? Our investigation of a set of other mammalian cystatin superfamily members indicated that the capacity to inhibit legumain is a property of only some cystatins (Table II). Guided by this result and amino acid sequence comparisons, we propose that the side of cystatins directly opposite to the papain-binding surface is responsible for the legumain binding and inhibition. The loop segment connecting the main α-helix of the cystatin structure to the first long β-strand contains a conserved Asn residue (residue 39 in cystatin C) and seems quite conserved in sequence in those cystatins that show inhibitory activity: cystatins C, E/M, and F. An importance of the Asn39 residue was confirmed by construction of the N39K cystatin C variant, which was seen to lack legumain inhibitory activity. A correctly positioned Asn residue on the cystatin surface could possibly result in an initial substrate-like interaction between the inhibitor and legumain. Besides the requirement for an Asn residue in the P1 position, legumains have no clear preferences for residues in other subsites (17Dando P.M. Fortunato M. Smith L. Knight C.G. McKendrick J.E. Barrett A.J. Biochem. J. 1999; 339: 743-749Crossref PubMed Scopus (61) Google Scholar,54Abe Y. Shirane K. Yokosawa H. Matsushita H. Mitta M. Kato I. Ishii S. J. Biol. Chem. 1993; 268: 3525-3529Abstract Full Text PDF PubMed Google Scholar). There are therefore few obvious structural possibilities for specific legumain inhibition besides interaction with the S1 pocket. Still, assuming that the "back-side loop" containing Asn39 interacts with the enzyme in a mode resembling substrate binding, it appeared from the inhibition data obtained that a loop segment preferentially containing polar amino acids is compatible with legumain interaction. The consensus sequence found in the three inhibitory cystatins is Ser(Thr)-Asn39-Asp(Ser)-Met(Ile). Strikingly, a Ser38-Asn39-Asp40 sequence is completely conserved in mouse, rat, and bovine cystatin C, as well as in chicken cystatin, which also inhibits pig legumain (15Chen J.-M. Dando P.M. Rawlings N.D. Brown M.A. Young N.E. Stevens R.A. Hewitt E. Watts C. Barrett A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). The positively charged Lys residue in this segment, present in the non-inhibitory cystatins B and D, may be unfavorable for inhibition.The suggested binding loop must be able to adopt a conformation to allow legumain interaction, but at the same time not expose the Asn39-Xaa bond to cleavage. A different back-side loop conformation may be one reason why the type 1 cystatins studied, with a loop sequence largely containing the proposed consensus sequence for legumain inhibition, although being two residues smaller, do not show inhibitory activity (Fig. 6). In the case of the inhibitory cystatins, the loop might be partially restrained in cystatin F as Cys37 likely is involved in a disulfide bridge (5Ni J. Fernandez M.A. Danielsson L. Chillakuru R.A. Zhang J. Grubb A. Su J. Gentz R. Abrahamson M. J. Biol. Chem. 1998; 273: 24797-24804Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), which can explain why cystatin F is a poorer inhibitor than cystatin C and E/M. The size and conformation of the loop could also be one reason why cystatin D does not inhibit legumain, because of an amino acid insertion in this loop (Fig. 4). For the type 3 cystatin studied, human L-kininogen, the lack of legumain-inhibitory activity may be due to steric reasons, as both legumain and kininogen are bulky molecules. Two of the three cystatin domains of kininogens are clearly able to inhibit papain-like peptidases (55Salvesen G. Parkes C. Abrahamson M. Grubb A. Barrett A.J. Biochem. J. 1986; 234: 429-434Crossref PubMed Scopus (176) Google Scholar), which demonstrates that the papain-binding surfaces of these domains are exposed and accessible to protein interactions. Whether the kininogen structure is sufficiently flexible to also allow exposure of the back-side loops on the opposite sides of these domains is presently unclear, as a three-dimensional model for type 3 cystatins is unfortunately not yet available. For the individual kininogen domains, the sequence requirements for a legumain-binding back-side loop suggested above seem to be fulfilled for domain 3, but not for domain 2 of human kininogen. Clearly, more studies are needed to clarify whether perhaps some variants of low or high M r kininogens, resulting from proteolytic cleavages to release the kinin portion or individual cystatin domains of the protein, display legumain-inhibitory activity.Although our initial studies indicate that the back-side loop around Asn39 is important for the ability of some cystatins to efficiently inhibit legumain, other cystatin segments may also be involved in interactions with the enzyme, just as several segments are involved in the cystatin inhibition of papain. The very flexible loop between the second and third of the four main β-strands of the cystatin structure, from Thr74 to Asn82 (which is not present in type 1 cystatins) may prove essential to stabilize the enzyme-inhibitor interaction, given its close proximity to the Asn39 loop (Fig. 5). Interestingly, this segment contains a five-residue insertion in the most efficient legumain inhibitor we identified, cystatin E/M (4Ni J. Abrahamson M. Zhang M. Fernandez M.A. Grubb A. Su J. Yu G.-L. Li Y. Parmelee D. Xing L. Coleman T.A. Gentz S. Thotakura R. Nguyen N. Hesselberg M. Gentz R. J. Biol. Chem. 1997; 272: 10853-10858Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 6Sotiropoulou G. Anisowicz A. Sager R. J. Biol. Chem. 1997; 272: 903-910Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). That this loop contains the primary binding site for legumain seems quite unlikely, however, as the loop sequence is relatively conserved between human cystatins C and D (Fig.4), of which only cystatin C shows legumain-inhibitory activity.In conclusion, our present results strongly indicate that the loop between the α-helix and the first strand of the main β-pleated sheet of the cystatin structure and its Asn39 residue, is part of a novel second reactive site of some cystatins. Cystatins carrying this site are sufficiently potent to be physiological inhibitors of mammalian legumain. Since legumain-like activity has very recently been shown to be crucial for cellular presentation of certain antigens to the immune system, but no efficient inhibitors to this activity are presently known (18Manoury B. Hewitt E.C. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (303) Google Scholar), continued studies to elucidate and explore the mechanism of legumain inhibition by the novel cystatin site may prove valuable. The activities of cysteine peptidases of the papain family (C1) such as cathepsins B, H, L, S, and K in and around mammalian cells are regulated by reversible, tight-binding protein inhibitors of the cystatin superfamily (1Abrahamson M. Methods Enzymol. 1994; 244: 685-700Crossref PubMed Scopus (182) Google Scholar). The cystatins constitute a superfamily of evolutionarily related proteins that are all composed of at least one 100–120-residue domain with conserved sequence motifs (2Rawlings N.D. Barrett A.J. J. Mol. Evol. 1990; 30: 60-71Crossref PubMed Scopus (267) Google Scholar). The single-domain human members of this superfamily are of two major types. The type 1 cystatins (or stefins) A and B contain approximately 100 amino acid residues, lack disulfide bridges, and are synthesized without signal peptides. Cystatins of type 2 are secreted proteins of approximately 120 amino acid residues (M r13,000–14,000) and contain at least two characteristic intrachain disulfide bonds. The type 2 cystatins include the human cystatins C, D, S, SN, and SA, which are all products of genes located in the cystatin multigene locus on chromosome 20 (3Schnittger S. Rao V.V. Abrahamson M. Hansmann I. Genomics. 1993; 16: 50-55Crossref PubMed Scopus (64) Google Scholar). Two recently identified type 2 cystatins, cystatin E/M and cystatin F (also called leukocystatin), are also secreted low M r proteins but are more atypical in that they are glycoproteins and show only 30–35% sequence identity in alignments with the classical type 2 cystatins. They are, however, still functional inhibitors of family C1 cysteine peptidases (4Ni J. Abrahamson M. Zhang M. Fernandez M.A. Grubb A. Su J. Yu G.-L. Li Y. Parmelee D. Xing L. Coleman T.A. Gentz S. Thotakura R. Nguyen N. Hesselberg M. Gentz R. J. Biol. Chem. 1997; 272: 10853-10858Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 5Ni J. Fernandez M.A. Danielsson L. Chillakuru R.A. Zhang J. Grubb A. Su J. Gentz R. Abrahamson M. J. Biol. Chem. 1998; 273: 24797-24804Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 6Sotiropoulou G. Anisowicz A. Sager R. J. Biol. Chem. 1997; 272: 903-910Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 7Halfon S. Ford J. Foster J. Dowling L. Lucian L. Sterling M. Xu Y. Weiss M. Ikeda M. Liggett D. Helms A. Caux C. Lebecque S. Hannum C. Menon S. McClanahan T. Gorman D. Zurawski G. J. Biol. Chem. 1998; 273: 16400-16408Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). It has been shown that the cystatin inhibition of cysteine peptidases of the papain family is due to a tripartite wedge-shaped structure with very good complementarity to the active site clefts of such enzymes (8Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar). The three parts of the cystatin polypeptide chain included in the enzyme-binding domain are the N-terminal segment, a central loop-forming segment with the motif Gln-Xaa-Val-Xaa-Gly, and a second C-terminal hairpin loop typically containing a Pro-Trp pair (8Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar, 9Abrahamson M. Ritonja A. Brown M.A. Grubb A. Machleidt W. Barrett A.J. J. Biol. Chem. 1987; 262: 9688-9694Abstract Full Text PDF PubMed Google Scholar, 10Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar). Legumain (EC 3.4.22.34) is a cysteine endopeptidase that was until recently known only from plants (11Kembhavi A.A. Buttle D.J. Knight C.G. Barrett A.J. Arch. Biochem. Biophys. 1993; 303: 208-213Crossref PubMed Scopus (158) Google Scholar, 12Hara-Nishimura I. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 746-749Google Scholar) and Schistosoma(13Dalton J.P. Brindley P.J. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 749-754Google Scholar). In plants there is abundant evidence that legumain performs a protein-processing function, causing limited proteolysis of precursor proteins and protein splicing (12Hara-Nishimura I. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 746-749Google Scholar, 14Min W. Jones H. Struct. Biology. 1994; 1: 502-504Crossref Scopus (58) Google Scholar). Following the discovery of the enzyme in mammalian cells, it was cloned and sequenced from human (15Chen J.-M. Dando P.M. Rawlings N.D. Brown M.A. Young N.E. Stevens R.A. Hewitt E. Watts C. Barrett A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar) and mouse (16Chen J.M. Dando P.M. Stevens R.A.E. Fortunato M. Barrett A.J. Biochem. J. 1998; 335: 111-117Crossref PubMed Scopus (112) Google Scholar). The amino acid sequences of legumains show that they belong to a distinct family of cysteine endopeptidases (C13). Mammalian legumain is predominantly lysosomal in distribution (16Chen J.M. Dando P.M. Stevens R.A.E. Fortunato M. Barrett A.J. Biochem. J. 1998; 335: 111-117Crossref PubMed Scopus (112) Google Scholar), but its strict specificity for the hydrolysis of bonds on the carboxyl side of asparagine is very different from that of any cathepsin and adapts it particularly for limited proteolysis (17Dando P.M. Fortunato M. Smith L. Knight C.G. McKendrick J.E. Barrett A.J. Biochem. J. 1999; 339: 743-749Crossref PubMed Scopus (61) Google Scholar). Human legumain may have an important physiological function as a key enzyme in antigen presentation (18Manoury B. Hewitt E.C. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (303) Google Scholar). It was recently reported that pig legumain is inhibited by human cystatin C and chicken cystatin with K i values below 5 nm (15Chen J.-M. Dando P.M. Rawlings N.D. Brown M.A. Young N.E. Stevens R.A. Hewitt E. Watts C. Barrett A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). This finding was unexpected, since the cystatins are already known as potent inhibitors of the papain-like cysteine peptidases in the unrelated family C1. The legumain family members are believed to have a protein fold quite unlike that of papain, and to be much more closely related to the caspases and gingipain (19Chen J.-M. Rawlings N.D. Stevens R.A.W. Barrett A.J. FEBS Lett. 1998; 441: 361-365Crossref PubMed Scopus (194) Google Scholar). Although the active site cysteine residue could seem to be a common factor, it is known not to be required for the interaction of papain with cystatins (20Björk I. Ylinenjarvi K. Biochem. J. 1989; 260: 61-68Crossref PubMed Scopus (37) Google Scholar). The present investigation was undertaken to elucidate the mechanism of inhibition of mammalian legumain by cystatins. DISCUSSIONThe aim of the present investigation was to study the mechanism of inhibition of mammalian legumain by cystatins, to clarify how the inhibitor structure can result in tight-binding inhibition of enzymes belonging to two entirely different enzyme families, namely the papain family (C1) and the legumain family (C13). The different arrangements of catalytic residues and different active site motifs show that the two families are evolutionarily unrelated, and that their peptidases have different protein folds (19Chen J.-M. Rawlings N.D. Stevens R.A.W. Barrett A.J. FEBS Lett. 1998; 441: 361-365Crossref PubMed Scopus (194) Google Scholar). Moreover, legumain is not inhibited by the general inhibitor of enzymes belonging to family C1, E-64 (15Chen J.-M. Dando P.M. Rawlings N.D. Brown M.A. Young N.E. Stevens R.A. Hewitt E. Watts C. Barrett A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar), which supports the theory that the general topography of the active site clefts of legumains are probably entirely different from those of family C1 enzymes. Our present SEC, electrophoretic and enzyme kinetic results show that cystatin C can inhibit mammalian legumain, as cystatins inhibit family C1 enzymes (52Nicklin M.J.H. Barrett A.J. Biochem. J. 1984; 223: 245-253Crossref PubMed Scopus (141) Google Scholar), by high affinity reversible binding (K i 0.20 nm), in a bimolecular reaction that is competitive with substrate, and with no detectable cleavage of the cystatin in the legumain complex. Despite these similarities, our present results demonstrate that the mechanisms of inhibition of legumain and family C1 endopeptidases must be completely different.From structural studies of several cystatins (8Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar, 9Abrahamson M. Ritonja A. Brown M.A. Grubb A. Machleidt W. Barrett A.J. J. Biol. Chem. 1987; 262: 9688-9694Abstract Full Text PDF PubMed Google Scholar, 10Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar), it is well known that the N-terminal segment together with the "first and second hairpin loops" in cystatins are responsible for the inhibition of the C1 enzymes (Fig. 5). Consequently, removal of the N-terminal segment or substitution of any of the conserved amino acids in the N-terminal segment or the hairpin loops by Gly/Ala residues abolishes or seriously affects the inhibition of papain-like enzymes (26Hall A. Håkansson K. Mason R.W. Grubb A. Abrahamson M. J. Biol. Chem. 1995; 270: 5115-5121Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 53Auerswald E.A. Genenger G. Assfalg-Machleidt I. Machleidt W. Engh R.A. Fritz H. Eur. J. Biochem. 1992; 209: 837-845Crossref PubMed Scopus (50) Google Scholar). Of four such variants analyzed in the present study, all displayed virtually unaltered binding of legumain. Dimeric cystatin C, which is completely inactive against papain-like enzymes and by NMR studies has been shown to be a result of intermolecular interactions between the papain-binding surfaces of two cystatin C molecules (47Ekiel I. Abrahamson M. Fulton D.B. Lindahl P. Storer A.C. Levadoux W. Lafrance M. Labelle S. Pomerleau Y. Groleau D. LeSauteur L. Gehring K. J. Mol. Biol. 1997; 271: 266-277Crossref PubMed Scopus (99) Google Scholar), still showed legumain inhibition. We believe that this, together with the enzyme kinetic results presented and the direct demonstration of a ternary complex by SEC, proves that the binding sites for papain and legumain on cystatin C likely are completely independent of each other.Where then is the legumain reactive site? Our investigation of a set of other mammalian cystatin superfamily members indicated that the capacity to inhibit legumain is a property of only some cystatins (Table II). Guided by this result and amino acid sequence comparisons, we propose that the side of cystatins directly opposite to the papain-binding surface is responsible for the legumain binding and inhibition. The loop segment connecting the main α-helix of the cystatin structure to the first long β-strand contains a conserved Asn residue (residue 39 in cystatin C) and seems quite conserved in sequence in those cystatins that show inhibitory activity: cystatins C, E/M, and F. An importance of the Asn39 residue was confirmed by construction of the N39K cystatin C variant, which was seen to lack legumain inhibitory activity. A correctly positioned Asn residue on the cystatin surface could possibly result in an initial substrate-like interaction between the inhibitor and legumain. Besides the requirement for an Asn residue in the P1 position, legumains have no clear preferences for residues in other subsites (17Dando P.M. Fortunato M. Smith L. Knight C.G. McKendrick J.E. Barrett A.J. Biochem. J. 1999; 339: 743-749Crossref PubMed Scopus (61) Google Scholar,54Abe Y. Shirane K. Yokosawa H. Matsushita H. Mitta M. Kato I. Ishii S. J. Biol. Chem. 1993; 268: 3525-3529Abstract Full Text PDF PubMed Google Scholar). There are therefore few obvious structural possibilities for specific legumain inhibition besides interaction with the S1 pocket. Still, assuming that the "back-side loop" containing Asn39 interacts with the enzyme in a mode resembling substrate binding, it appeared from the inhibition data obtained that a loop segment preferentially containing polar amino acids is compatible with legumain interaction. The consensus sequence found in the three inhibitory cystatins is Ser(Thr)-Asn39-Asp(Ser)-Met(Ile). Strikingly, a Ser38-Asn39-Asp40 sequence is completely conserved in mouse, rat, and bovine cystatin C, as well as in chicken cystatin, which also inhibits pig legumain (15Chen J.-M. Dando P.M. Rawlings N.D. Brown M.A. Young N.E. Stevens R.A. Hewitt E. Watts C. Barrett A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). The positively charged Lys residue in this segment, present in the non-inhibitory cystatins B and D, may be unfavorable for inhibition.The suggested binding loop must be able to adopt a conformation to allow legumain interaction, but at the same time not expose the Asn39-Xaa bond to cleavage. A different back-side loop conformation may be one reason why the type 1 cystatins studied, with a loop sequence largely containing the proposed consensus sequence for legumain inhibition, although being two residues smaller, do not show inhibitory activity (Fig. 6). In the case of the inhibitory cystatins, the loop might be partially restrained in cystatin F as Cys37 likely is involved in a disulfide bridge (5Ni J. Fernandez M.A. Danielsson L. Chillakuru R.A. Zhang J. Grubb A. Su J. Gentz R. Abrahamson M. J. Biol. Chem. 1998; 273: 24797-24804Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), which can explain why cystatin F is a poorer inhibitor than cystatin C and E/M. The size and conformation of the loop could also be one reason why cystatin D does not inhibit legumain, because of an amino acid insertion in this loop (Fig. 4). For the type 3 cystatin studied, human L-kininogen, the lack of legumain-inhibitory activity may be due to steric reasons, as both legumain and kininogen are bulky molecules. Two of the three cystatin domains of kininogens are clearly able to inhibit papain-like peptidases (55Salvesen G. Parkes C. Abrahamson M. Grubb A. Barrett A.J. Biochem. J. 1986; 234: 429-434Crossref PubMed Scopus (176) Google Scholar), which demonstrates that the papain-binding surfaces of these domains are exposed and accessible to protein interactions. Whether the kininogen structure is sufficiently flexible to also allow exposure of the back-side loops on the opposite sides of these domains is presently unclear, as a three-dimensional model for type 3 cystatins is unfortunately not yet available. For the individual kininogen domains, the sequence requirements for a legumain-binding back-side loop suggested above seem to be fulfilled for domain 3, but not for domain 2 of human kininogen. Clearly, more studies are needed to clarify whether perhaps some variants of low or high M r kininogens, resulting from proteolytic cleavages to release the kinin portion or individual cystatin domains of the protein, display legumain-inhibitory activity.Although our initial studies indicate that the back-side loop around Asn39 is important for the ability of some cystatins to efficiently inhibit legumain, other cystatin segments may also be involved in interactions with the enzyme, just as several segments are involved in the cystatin inhibition of papain. The very flexible loop between the second and third of the four main β-strands of the cystatin structure, from Thr74 to Asn82 (which is not present in type 1 cystatins) may prove essential to stabilize the enzyme-inhibitor interaction, given its close proximity to the Asn39 loop (Fig. 5). Interestingly, this segment contains a five-residue insertion in the most efficient legumain inhibitor we identified, cystatin E/M (4Ni J. Abrahamson M. Zhang M. Fernandez M.A. Grubb A. Su J. Yu G.-L. Li Y. Parmelee D. Xing L. Coleman T.A. Gentz S. Thotakura R. Nguyen N. Hesselberg M. Gentz R. J. Biol. Chem. 1997; 272: 10853-10858Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 6Sotiropoulou G. Anisowicz A. Sager R. J. Biol. Chem. 1997; 272: 903-910Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). That this loop contains the primary binding site for legumain seems quite unlikely, however, as the loop sequence is relatively conserved between human cystatins C and D (Fig.4), of which only cystatin C shows legumain-inhibitory activity.In conclusion, our present results strongly indicate that the loop between the α-helix and the first strand of the main β-pleated sheet of the cystatin structure and its Asn39 residue, is part of a novel second reactive site of some cystatins. Cystatins carrying this site are sufficiently potent to be physiological inhibitors of mammalian legumain. Since legumain-like activity has very recently been shown to be crucial for cellular presentation of certain antigens to the immune system, but no efficient inhibitors to this activity are presently known (18Manoury B. Hewitt E.C. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (303) Google Scholar), continued studies to elucidate and explore the mechanism of legumain inhibition by the novel cystatin site may prove valuable. The aim of the present investigation was to study the mechanism of inhibition of mammalian legumain by cystatins, to clarify how the inhibitor structure can result in tight-binding inhibition of enzymes belonging to two entirely different enzyme families, namely the papain family (C1) and the legumain family (C13). The different arrangements of catalytic residues and different active site motifs show that the two families are evolutionarily unrelated, and that their peptidases have different protein folds (19Chen J.-M. Rawlings N.D. Stevens R.A.W. Barrett A.J. FEBS Lett. 1998; 441: 361-365Crossref PubMed Scopus (194) Google Scholar). Moreover, legumain is not inhibited by the general inhibitor of enzymes belonging to family C1, E-64 (15Chen J.-M. Dando P.M. Rawlings N.D. Brown M.A. Young N.E. Stevens R.A. Hewitt E. Watts C. Barrett A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar), which supports the theory that the general topography of the active site clefts of legumains are probably entirely different from those of family C1 enzymes. Our present SEC, electrophoretic and enzyme kinetic results show that cystatin C can inhibit mammalian legumain, as cystatins inhibit family C1 enzymes (52Nicklin M.J.H. Barrett A.J. Biochem. J. 1984; 223: 245-253Crossref PubMed Scopus (141) Google Scholar), by high affinity reversible binding (K i 0.20 nm), in a bimolecular reaction that is competitive with substrate, and with no detectable cleavage of the cystatin in the legumain complex. Despite these similarities, our present results demonstrate that the mechanisms of inhibition of legumain and family C1 endopeptidases must be completely different. From structural studies of several cystatins (8Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (543) Google Scholar, 9Abrahamson M. Ritonja A. Brown M.A. Grubb A. Machleidt W. Barrett A.J. J. Biol. Chem. 1987; 262: 9688-9694Abstract Full Text PDF PubMed Google Scholar, 10Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (463) Google Scholar), it is well known that the N-terminal segment together with the "first and second hairpin loops" in cystatins are responsible for the inhibition of the C1 enzymes (Fig. 5). Consequently, removal of the N-terminal segment or substitution of any of the conserved amino acids in the N-terminal segment or the hairpin loops by Gly/Ala residues abolishes or seriously affects the inhibition of papain-like enzymes (26Hall A. Håkansson K. Mason R.W. Grubb A. Abrahamson M. J. Biol. Chem. 1995; 270: 5115-5121Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 53Auerswald E.A. Genenger G. Assfalg-Machleidt I. Machleidt W. Engh R.A. Fritz H. Eur. J. Biochem. 1992; 209: 837-845Crossref PubMed Scopus (50) Google Scholar). Of four such variants analyzed in the present study, all displayed virtually unaltered binding of legumain. Dimeric cystatin C, which is completely inactive against papain-like enzymes and by NMR studies has been shown to be a result of intermolecular interactions between the papain-binding surfaces of two cystatin C molecules (47Ekiel I. Abrahamson M. Fulton D.B. Lindahl P. Storer A.C. Levadoux W. Lafrance M. Labelle S. Pomerleau Y. Groleau D. LeSauteur L. Gehring K. J. Mol. Biol. 1997; 271: 266-277Crossref PubMed Scopus (99) Google Scholar), still showed legumain inhibition. We believe that this, together with the enzyme kinetic results presented and the direct demonstration of a ternary complex by SEC, proves that the binding sites for papain and legumain on cystatin C likely are completely independent of each other. Where then is the legumain reactive site? Our investigation of a set of other mammalian cystatin superfamily members indicated that the capacity to inhibit legumain is a property of only some cystatins (Table II). Guided by this result and amino acid sequence comparisons, we propose that the side of cystatins directly opposite to the papain-binding surface is responsible for the legumain binding and inhibition. The loop segment connecting the main α-helix of the cystatin structure to the first long β-strand contains a conserved Asn residue (residue 39 in cystatin C) and seems quite conserved in sequence in those cystatins that show inhibitory activity: cystatins C, E/M, and F. An importance of the Asn39 residue was confirmed by construction of the N39K cystatin C variant, which was seen to lack legumain inhibitory activity. A correctly positioned Asn residue on the cystatin surface could possibly result in an initial substrate-like interaction between the inhibitor and legumain. Besides the requirement for an Asn residue in the P1 position, legumains have no clear preferences for residues in other subsites (17Dando P.M. Fortunato M. Smith L. Knight C.G. McKendrick J.E. Barrett A.J. Biochem. J. 1999; 339: 743-749Crossref PubMed Scopus (61) Google Scholar,54Abe Y. Shirane K. Yokosawa H. Matsushita H. Mitta M. Kato I. Ishii S. J. Biol. Chem. 1993; 268: 3525-3529Abstract Full Text PDF PubMed Google Scholar). There are therefore few obvious structural possibilities for specific legumain inhibition besides interaction with the S1 pocket. Still, assuming that the "back-side loop" containing Asn39 interacts with the enzyme in a mode resembling substrate binding, it appeared from the inhibition data obtained that a loop segment preferentially containing polar amino acids is compatible with legumain interaction. The consensus sequence found in the three inhibitory cystatins is Ser(Thr)-Asn39-Asp(Ser)-Met(Ile). Strikingly, a Ser38-Asn39-Asp40 sequence is completely conserved in mouse, rat, and bovine cystatin C, as well as in chicken cystatin, which also inhibits pig legumain (15Chen J.-M. Dando P.M. Rawlings N.D. Brown M.A. Young N.E. Stevens R.A. Hewitt E. Watts C. Barrett A.J. J. Biol. Chem. 1997; 272: 8090-8098Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). The positively charged Lys residue in this segment, present in the non-inhibitory cystatins B and D, may be unfavorable for inhibition. The suggested binding loop must be able to adopt a conformation to allow legumain interaction, but at the same time not expose the Asn39-Xaa bond to cleavage. A different back-side loop conformation may be one reason why the type 1 cystatins studied, with a loop sequence largely containing the proposed consensus sequence for legumain inhibition, although being two residues smaller, do not show inhibitory activity (Fig. 6). In the case of the inhibitory cystatins, the loop might be partially restrained in cystatin F as Cys37 likely is involved in a disulfide bridge (5Ni J. Fernandez M.A. Danielsson L. Chillakuru R.A. Zhang J. Grubb A. Su J. Gentz R. Abrahamson M. J. Biol. Chem. 1998; 273: 24797-24804Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), which can explain why cystatin F is a poorer inhibitor than cystatin C and E/M. The size and conformation of the loop could also be one reason why cystatin D does not inhibit legumain, because of an amino acid insertion in this loop (Fig. 4). For the type 3 cystatin studied, human L-kininogen, the lack of legumain-inhibitory activity may be due to steric reasons, as both legumain and kininogen are bulky molecules. Two of the three cystatin domains of kininogens are clearly able to inhibit papain-like peptidases (55Salvesen G. Parkes C. Abrahamson M. Grubb A. Barrett A.J. Biochem. J. 1986; 234: 429-434Crossref PubMed Scopus (176) Google Scholar), which demonstrates that the papain-binding surfaces of these domains are exposed and accessible to protein interactions. Whether the kininogen structure is sufficiently flexible to also allow exposure of the back-side loops on the opposite sides of these domains is presently unclear, as a three-dimensional model for type 3 cystatins is unfortunately not yet available. For the individual kininogen domains, the sequence requirements for a legumain-binding back-side loop suggested above seem to be fulfilled for domain 3, but not for domain 2 of human kininogen. Clearly, more studies are needed to clarify whether perhaps some variants of low or high M r kininogens, resulting from proteolytic cleavages to release the kinin portion or individual cystatin domains of the protein, display legumain-inhibitory activity. Although our initial studies indicate that the back-side loop around Asn39 is important for the ability of some cystatins to efficiently inhibit legumain, other cystatin segments may also be involved in interactions with the enzyme, just as several segments are involved in the cystatin inhibition of papain. The very flexible loop between the second and third of the four main β-strands of the cystatin structure, from Thr74 to Asn82 (which is not present in type 1 cystatins) may prove essential to stabilize the enzyme-inhibitor interaction, given its close proximity to the Asn39 loop (Fig. 5). Interestingly, this segment contains a five-residue insertion in the most efficient legumain inhibitor we identified, cystatin E/M (4Ni J. Abrahamson M. Zhang M. Fernandez M.A. Grubb A. Su J. Yu G.-L. Li Y. Parmelee D. Xing L. Coleman T.A. Gentz S. Thotakura R. Nguyen N. Hesselberg M. Gentz R. J. Biol. Chem. 1997; 272: 10853-10858Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 6Sotiropoulou G. Anisowicz A. Sager R. J. Biol. Chem. 1997; 272: 903-910Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). That this loop contains the primary binding site for legumain seems quite unlikely, however, as the loop sequence is relatively conserved between human cystatins C and D (Fig.4), of which only cystatin C shows legumain-inhibitory activity. In conclusion, our present results strongly indicate that the loop between the α-helix and the first strand of the main β-pleated sheet of the cystatin structure and its Asn39 residue, is part of a novel second reactive site of some cystatins. Cystatins carrying this site are sufficiently potent to be physiological inhibitors of mammalian legumain. Since legumain-like activity has very recently been shown to be crucial for cellular presentation of certain antigens to the immune system, but no efficient inhibitors to this activity are presently known (18Manoury B. Hewitt E.C. Morrice N. Dando P.M. Barrett A.J. Watts C. Nature. 1998; 396: 695-699Crossref PubMed Scopus (303) Google Scholar), continued studies to elucidate and explore the mechanism of legumain inhibition by the novel cystatin site may prove valuable. We gratefully acknowledge the skilled technical assistance of Anne-Cathrine Löfström, Inger Nilsson, and Lorraine Smith. We thank Drs. Ingemar Björk and Anders Grubb for support and gifts of cystatin preparations used to generate the data presented in Table II.
Referência(s)