Cystatin S: a cysteine proteinase inhibitor of human saliva

An acidic protein of human saliva, which we named SAP-1 previously, is now shown to be an inhibitor of several cysteine proteinases. The protein inhibited papain and ficin strongly, and stem bromelain and bovine cathepsin C partially. However, it did not inhibit either porcine cathepsin B or clostripain. The mode of the inhibition of papain was found to be non-competitive. The name cystatin S has been proposed for this salivary protein in view of the similarities in activity and structure to other cysteine proteinase inhibitors such as chicken egg-white cystatin and human cystatins A, B, and C. The cystatin S antigen was detected immunohistochemically in the serous cells of human parotid and submaxillary glands.     

Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin

When an excess of human cystatin C or chicken cystatin was mixed with papain, an enzyme-inhibitor complex was formed immediately. The residual free cystatin was then progressively converted to a form with different electrophoretic mobility and chromatographic properties. The modified cystatins were isolated and sequenced, showing that there had been cleavage of a single peptide bond in each molecule: Gly11-Gly12 in cystatin C, and Gly9-Ala10 in chicken cystatin. The residues Gly11 (cystatin C) and Gly9 (chicken cystatin) are among only three residues conserved in all known sequences of inhibitory cystatins. The modified cystatins were at least 1000-fold weaker inhibitors of papain than the native cystatins. An 18-residue synthetic peptide corresponding to residues 4-21 of cystatin C did not inhibit papain but was cleaved at the same Gly-Gly bond as cystatin C. When iodoacetate or L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane was added to the mixtures of either cystatin with papain, modification of the excess cystatin was blocked. Papain-cystatin complexes were stable to prolonged incubation, even in the presence of excess papain. We conclude that the peptidyl bond of the conserved glycine residue in human cystatin C and chicken cystatin probably is part of a substrate-like inhibitory reactive site of these cysteine proteinase inhibitors of the cystatin superfamily and that this may be true also for other inhibitors of this superfamily. We also propose that human cystatin C and chicken cystatin, and probably other cystatins as well, inhibit cysteine proteinases by the simultaneous interactions with such proteinases of the inhibitory reactive sites and other, so far not identified, areas of the cystatins. The cleavage of the inhibitory reactive site glycyl bond in mixtures of papain with excess quantities of cystatins is apparently due to the activity of a small percentage of atypical cysteine proteinase molecules in the papain preparation that form only very loose complexes with cystatins under the conditions employed and degrade the free cystatin molecules.     

Rat tissue concentrations of branched-chain 2-oxo acid dehydrogenase complex. Re-evaluation by immunoassay and bioassay.

We point out that human low-Mr kininogen contains three cystatin-like sequences, rather than two, as had previously been thought. The protein was purified by affinity chromatography on carboxymethyl-papain-Sepharose, and subjected to limited proteolysis by trypsin and chymotrypsin. Fragments were isolated, and three corresponding to the individual cystatin-like domains were identified. By comparison with the known amino acid sequence of the protein they were numbered 1 to 3 from the N-terminus. Domain 1 was not found to have any inhibitory activity for cysteine proteinases, which is consistent with the absence of residues that are highly conserved in inhibitors of the cystatin superfamily, and have previously been suggested to be essential for activity. Domain 2 was a good inhibitor of chicken calpain, and also papain and cathepsin L. Domain 3 showed negligible inhibition of calpain, but inhibited papain and cathepsin L strongly. The probable arrangement of disulphide bonds in the heavy chain of low-Mr kininogen is deduced from the homology with the cystatins and other evidence contained in the present paper.     

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 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.     

Synthetic domains of cystatins linked to enkephalins are novel inhibitors of brain cathepsins L/B

Cystatin domains or homologous sequences were synthesized and tested as inhibitors of papain, and rat brain cathepsins B and L. These domains included: I, an enzyme substrate binding site containing a -GG- cleavage site (YGGFL); II, known cystatin consensus sequences (-QVVAG- or -QLVSG-); and III, the proposed ancillary site for binding of chicken cystatin to papain (-IPWLN-). A Domain II analog QVVAG(K-NH2) inhibited cathepsin L and papain with Ki 1-4 X 10(-4) M but was inactive towards cathepsin B. A peptide containing Domains I and II, YGGFL-QVVAG(K-NH2), inhibited papain and cathepsin B with Ki 10(-4)-10(-5) M, and cathepsin L with Ki 10(-6) M. The presence of Domain III in the analog YGGFL-QVVAG-IPWLN(K-NH2) resulted in a 10-fold increase in potency towards papain. These data demonstrated that putative cystatin domains are: 1) probably proximal in the intact cystatins; 2) can be linked directly to each other to yield smaller peptides active as inhibitors; 3) showed some specificity towards the three cysteine proteinases.     

Down-regulation of human extracellular cysteine protease inhibitors by the secreted staphylococcal cysteine proteases, staphopain A and B

Of seven human cystatins investigated, none inhibited the cysteine proteases staphopain A and B secreted by the human pathogen Staphylococcus aureus. Rather, the extracellular cystatins C, D and E/M were hydrolyzed by both staphopains. Based on MALDI-TOF time-course experiments, staphopain A cleavage of cystatin C and D should be physiologically relevant and occur upon S. aureus infection. Staphopain A hydrolyzed the Gly11 bond of cystatin C and the Ala10 bond of cystatin D with similar Km values of approximately 33 and 32 microM, respectively. Such N-terminal truncation of cystatin C caused >300-fold lower inhibition of papain, cathepsin B, L and K, whereas the cathepsin H activity was compromised by a factor of ca. 10. Similarly, truncation of cystatin D caused alleviated inhibition of all endogenous target enzymes investigated. The normal activity of the cystatins is thus down-regulated, indicating that the bacterial enzymes can cause disturbance of the host protease-inhibitor balance. To illustrate the in vivo consequences, a mixed cystatin C assay showed release of cathepsin B activity in the presence of staphopain A. Results presented for the specificity of staphopains when interacting with cystatins as natural protein substrates could aid in the development of therapeutic agents directed toward these proteolytic virulence factors.     

Cathepsin D inactivates cysteine proteinase inhibitors, cystatins

The formation of inactive complexes in excess molar amounts of human cathepsins H and L with their protein inhibitors human stefin A, human stefin B and chicken cystatin at pH 5.6 has been shown by measurement of enzyme activity coupled with reverse-phase HPLC not to involve covalent cleavage of the inhibitors. Inhibition must be the direct result of binding. On the contrary the interaction of cystatins with aspartic proteinase cathepsin D at pH 3.5 for 60 min followed by HPLC resulted in their inactivation accompanied by peptide bond cleavage at several sites, preferentially those involving hydrophobic amino acid residues. The released peptides do not inhibit papain and cathepsin L. These results explain reported elevated levels of cysteine proteinases and lead to the proposal that cathepsin D exerts an important function, through inactivation of cystatins, in the increased activities of cysteine proteinases in human diseases including muscular distrophy.     

The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors

Native gamma-trace, a small basic protein present in high concentration in cerebrospinal fluid, semen and neuroendocrine cells, but of unknown biological function, is shown to be a potent inhibitor of the cysteine proteinases papain, ficin, and human cathepsins B, H and L. It proves to be the tightest -binding protein inhibitor of cathepsin B so far discovered. The name cystatin C is proposed for gamma-trace to reflect the many similarities in activity and structure to chicken egg-white cystatin and mammalian cystatins A and B. The inhibition constants of cystatin C, taken together with its widespread distribution in human tissues and extracellular fluids, suggest that a physiological function could well be the regulation of cysteine proteinase activity.     

Internalization of cystatin C in human cell lines

Altered protease activity is considered important for tumour invasion and metastasis, processes in which the cysteine proteases cathepsin B and L are involved. Their natural inhibitor cystatin C is a secreted protein, suggesting that it functions to control extracellular protease activity. Because cystatins added to cell cultures can inhibit polio, herpes simplex and coronavirus replication, which are intracellular processes, the internalization and intracellular regulation of cysteine proteases by cystatin C should be considered. The extension, mechanism and biological importance of this hypothetical process are unknown. We investigated whether internalization of cystatin C occurs in a set of human cell lines. Demonstrated by flow cytometry and confocal microscopy, A-431, MCF-7, MDA-MB-453, MDA-MB-468 and Capan-1 cells internalized fluorophore-conjugated cystatin C when exposed to physiological concentrations (1 microm). During cystatin C incubation, intracellular cystatin C increased after 5 min and accumulated for at least 6 h, reaching four to six times the baseline level. Western blotting showed that the internalized inhibitor was not degraded. It was functionally intact and extracts of cells exposed to cystatin C showed a higher capacity to inhibit papain and cathepsin B than control cells (decrease in enzyme activity of 34% and 37%, respectively). The uptake of labelled cystatin C was inhibited by unlabelled inhibitor, suggesting a specific pathway for the internalization. We conclude that the cysteine protease inhibitor cystatin C is internalized in significant quantities in various cancer cell lines. This is a potentially important physiological phenomenon not previously described for this group of inhibitors.     

Inhibition of cysteine proteinases by a protein inhibitor from potato

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg-white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.     

Cystatins

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.     

Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases

Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by approximately 15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by approximately 10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering.     

Inhibitory properties of low molecular mass cysteine proteinase inhibitors from human sarcoma

Elevated activities of cysteine proteinases such as cathepsins B and L and cancer procoagulant have been linked to tumor malignancy. In the present study we examined the hypothesis that these elevated activities could be due to impaired regulation by the endogenous low molecular mass cysteine proteinase inhibitors (cystatins). Inhibitors from human sarcoma were compared to those from human liver, a normal tissue in which the inhibitors had been characterized previously. An extract of cystatins from sarcoma was less effective against papain and cathepsin B (liver or tumor) than was an extract from liver. This reduced inhibitory capacity in sarcoma was not due to a reduction in either the concentrations or specific activities of the cystatins or an absence of any family or isoform of cystatins. We purified two members of the cystatin superfamily (stefin A and stefin B) to homogeneity and determined their individual inhibitory properties. Stefins B from liver and sarcoma exhibited comparable inhibition of papain and cathepsin B. In contrast, stefin A from sarcoma exhibited a reduced ability to inhibit papain, human liver cathepsins B, H and L and human and murine tumor cathepsin B. The Ki for inhibition of liver cathepsin B by sarcoma stefin A was 10-fold higher than that for inhibition of liver cathepsin B by liver stefin A, reflecting a reduction in the rate constant for association and an increase in the rate constant for dissociation. Cancer is now the third pathologic condition reported to be associated with alterations in cystatins, the other two being amyloidosis and muscular dystrophy.     

Cystatins as calpain inhibitors: engineered chicken cystatin- and stefin B-kininogen domain 2 hybrids support a cystatin-like mode of interaction with the catalytic subunit of mu-calpain

Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit mu- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and deltaL110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of Ki = 188 nM. Deletion of L110, which forms a beta-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of mu-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the mu-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.     

Inhibition of plant-pathogenic fungi by the barley cystatin Hv-CPI (gene Icy) is not associated with its cysteine-proteinase inhibitory properties

The recombinant barley cystatin Hv-CPI inhibited the growth of three phytopathogenic fungi (Botrytis cinerea, Colletotrichum graminicola, and Plectosphaerella cucumerina) and the saprotrophic fungus Trichoderma viride. Several mutants of barley cystatin were generated by polymerase chain reaction approaches and both their antifungal and their cysteine-proteinase inhibitory properties investigated. Point mutants R38-->G, Q63-->L, and Q63-->P diminished their capacity for inhibiting papain and cathepsin B, retaining their antifungal properties. However, mutant C68-->G was more active for papain and cathepsin B than the wild type. These results indicate that in addition to the consensus cystatin-reactive site, Q63-V64-V65-A66-G67, the A37-R38-F39-A40-V41 region, common to all cereal cystatins, and the C68 residue are important for barley cystatin activity. On the other hand, the K92-->P mutant is inactive as a fungicide, but still retains measurable inhibitory activity for papain and cathepsin B. Against B. cinerea, the antifungal effect of Hv-CPI and of its derived mutants does not always correlate with their activities as proteinase inhibitors, because the Q63-->P mutant is inactive as a cystatin, while still inhibiting fungal growth, and the K92-->P mutant shows the reciprocal effects. These data indicate that inhibition of plant-pathogenic fungi by barley cystatin is not associated with its cysteine-proteinase inhibitory activity. Moreover, these results are corroborated by the absence of inhibition of intra- and extramycelia-proteinase activities by barley cystatin and by other well-known inhibitors of cysteine-proteinase activity in the fungal zymograms of B. cinerea.     

Identification of cysteine protease inhibitors that belong to cystatin family 1 in the cellular slime mold Dictyostelium discoideum

Family 1 cystatins are cytosolic inhibitors of cysteine proteases, and they are conserved in higher eukaryotes. We characterized two newly identified family 1 cystatins of the cellular slime mold Dictyostelium discoideum, cystatin A1 and A2. Their recombinant proteins showed specific inhibitory activity against papain and cathepsin B, respectively. Using specific polyclonal antibodies, we found that cystatin A1 is stably expressed throughout the life cycle of Dictyostelium, whereas cystatin A2 expression is up-regulated during the course of development.     

Three-dimensional solution structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica

The three-dimensional structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica, has been determined in solution at pH 6.8 and 25 degrees C by (1)H and (15)N NMR spectroscopy. The main body (Glu13-Asp97) of oryzacystatin-I is well-defined and consists of an alpha-helix and a five-stranded antiparallel beta-sheet, while the N- and C-terminal regions (Ser2-Val12 and Ala98-Ala102) are less defined. The helix-sheet architechture of oryzacystatin-I is stabilized by a hydrophobic cluster formed between the alpha-helix and the beta-sheet and is considerably similar to that of monellin, a sweet-tasting protein from an African berry, as well as those of the animal cystatins studied, e.g., chicken egg white cystatin and human stefins A and B (also referred to as human cystatins A and B). Detailed structural comparison indicates that oryzacystatin-I is more similar to chicken cystatin, which belongs to the type-2 animal cystatins, than to human stefins A and B, which belong to the type-1 animal cystatins, despite different loop length.     

Inhibition of chicken calpain II by proteins of the cystatin superfamily and alpha 2-macroglobulin.

Inhibition of chicken calpain II by proteins of the cystatin superfamily and alpha 2-macroglobulin was investigated. Human liver cystatins A and B, human cystatin C, chicken cystatin and rat T-kininogen were found not to be inhibitory. Inhibition was, however, observed for bovine and rat kininogens, with Ki (inhibition constant) values of 0.8 nM and 30 nM respectively. alpha 2-Macroglobulin inhibits calpain with an initial rate constant of the order of 3 X 10(4) M-1.S-1. Calpain complexed with alpha 2-macroglobulin showed only limited reactivity towards azocasein, but reacted readily with the peptide substrate Suc-Leu-Tyr-4-methyl-7-coumarylamide and with L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64). The calpain in the complexes was at least partially protected from loss of activity due to autolysis. The calpain-alpha 2-macroglobulin complexes contained both the calpain subunits.     

The N-terminal region of cystatin A (stefin A) binds to papain subsequent to the two hairpin loops of the inhibitor. Demonstration of two-step binding by rapid-kinetic studies of cystatin A labeled at the N-terminus with a fluorescent reporter group

The three-dimensional structures of cystatins, and other evidence, suggest that the flexible N-terminal region of these inhibitors may bind to target proteinases independent of the two rigid hairpin loops forming the remainder of the inhibitory surface. In an attempt to demonstrate such two-step binding, which could not be identified in previous kinetics studies, we introduced a cysteine residue before the N-terminus of cystatin A and labeled this residue with fluorescent probes. Binding of AANS- and AEDANS-labeled cystatin A to papain resulted in approximately 4-fold and 1.2-fold increases of probe fluorescence, respectively, reflecting the interaction of the N-terminal region with the enzyme. Observed pseudo-first-order rate constants, measured by the loss of papain activity in the presence of a fluorogenic substrate, for the reaction of the enzyme with excess AANS-cystatin A increased linearly with the concentration of the latter. In contrast, pseudo-first-order rate constants, obtained from measurements of the change of probe fluorescence with either excess enzyme or labeled inhibitor, showed an identical hyperbolic dependence on the concentration of the reactant in excess. This dependence demonstrates that the binding occurs in two steps, and implies that the labeled N-terminal region of cystatin A interacts with the proteinase in the second step, subsequent to the hairpin loops. The comparable affinities and dissociation rate constants for the binding of labeled and unlabeled cystatin A to papain indicate that the label did not appreciably perturb the interaction, and that unlabeled cystatin therefore also binds in a similar two-step manner. Such independent binding of the N-terminal regions of cystatins to target proteinases after the hairpin loops may be characteristic of most cystatin-proteinase reactions.     

Comparative Analysis of Cystatin Superfamily in Platyhelminths

The cystatin superfamily is comprised of cysteine proteinase inhibitors and encompasses at least 3 subfamilies: stefins, cystatins and kininogens. In this study, the platyhelminth cystatin superfamily was identified and grouped into stefin and cystatin subfamilies. The conserved domain of stefins (G, QxVxG) was observed in all members of platyhelminth stefins. The three characteristics of cystatins, the cystatin-like domain (G, QxVxG, PW), a signal peptide, and one or two conserved disulfide bonds, were observed in platyhelminths, with the exception of cestodes, which lacked the conserved disulfide bond. However, it is noteworthy that cestode cystatins had two tandem repeated domains, although the second tandem repeated domain did not contain a cystatin-like domain, which has not been previously reported. Tertiary structure analysis of Taenia solium cystatin, one of the cestode cystatins, demonstrated that the N-terminus of T. solium cystatin formed a five turn α-helix, a five stranded β-pleated sheet and a hydrophobic edge, similar to the structure of chicken cystatin. Although no conserved disulfide bond was found in T. solium cystatin, the models of T. solium cystatin and chicken cystatin corresponded at the site of the first disulfide bridge of the chicken cystatin. However, the two models were not similar regarding the location of the second disulfide bridge of chicken cystatin. These results showed that T. solium cystatin and chicken cystatin had similarities and differences, suggesting that the biochemistry of T. solium cystatin could be similar to chicken cystatin in its inhibitory function and that it may have further functional roles. The same results were obtained for other cestode cystatins. Phylogenetic analysis showed that cestode cystatins constituted an independent clade and implied that cestode cystatins should be considered to have formed a new clade during evolution.