Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin.

The interactions between egg-white cystatin and the cysteine proteinases papain, human cathepsin B and bovine dipeptidyl peptidase I were studied. Cystatin was shown to be a competitive reversible inhibitor of cathepsin B (Ki 1.7 nM, k-1 about 2.3 X 10(-3) s-1). The inhibition of dipeptidyl peptidase I was shown to be reversible (Ki(app.) 0.22 nM, k-1 about 2.2 X 10(-3) s-1). Cystatin bound papain too tightly for Ki to be determined, but an upper limit of 5 pM was estimated. The association was a second-order process, with k+1 1.0 X 10(7) M-1 X s-1. Papain was shown to form equimolar complexes with cystatin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of complexes formed between papain or cathepsin B and an excess of cystatin showed no peptide bond cleavage after incubation for 72 h. The reaction of the active-site thiol group of papain with 5,5'-dithiobis-(2-nitrobenzoic acid) at pH 8 and 2,2'-dithiobispyridine at pH 4 was blocked by complex-formation. Dipeptidyl peptidase I and papain were found to compete for binding to cystatin, contrary to a previous report. The two major isoelectric forms of cystatin were found to have similar specific inhibitory activities for papain, and similar affinities for papain, cathepsin B and dipeptidyl peptidase I. This, together with specific oxidation of the N-terminal serine residue with periodate, showed the N-terminal amino group of cystatin 1 to be unimportant for inhibition. General citraconylation of amino groups resulted in a large decrease in the affinity of cystatin for dipeptidyl peptidase I. It is concluded that the interaction of cystatin with cysteine proteinases has many characteristics similar to those of an inhibitor such as aprotinin with serine proteinases.     

Cystatin-like cysteine proteinase inhibitors from human liver.

Cysteine proteinase inhibitor (CPI) forms from human liver were purified from the tissue homogenate by alkaline denaturation of cysteine proteinases with which they are complexed, acetone fractionation, affinity chromatography on S-carboxymethyl-papain-Sepharose and chromatofocusing. The multiple forms of CPI were shown immunologically to be forms of two proteins, referred to as CPI-A (comprising the forms of relatively acidic pI) and CPI-B (comprising the more basic forms). CPI-A and CPI-B are similar in their Mr of about 12400, considerable stability to pH2, pH11 and 80 degrees C, and tight-binding inhibition of papain, several related cysteine proteinases and dipeptidyl peptidase I. Ki values were determined for papain, human cathepsins B, H and L, and dipeptidyl peptidase I. The affinity of CPI-A for cathepsin B was about 10-fold greater than that of CPI-B, whereas CBI-B showed about 100-fold stronger inhibition of dipeptidyl peptidase I. For all the cysteine proteinases the liver inhibitors were somewhat less tight binding than cystatin. The resemblance of both CPI-A and CPI-B in several respects to egg-white cystatin is discussed. CPI-A seems to correspond to the epithelial inhibitor described previously, and CPI-B to the inhibitor from other cell types [Järvinen & Rinne (1982) Biochim. Biophys. Acta 708, 210-217].     

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.     

Human cystatin, a new protein inhibitor of cysteine proteinases

A new low-molecular weight protein inhibitor of cysteine proteinases, human cystatin, was isolated from sera of patients with autoimmune diseases. It inhibits papain, human cathepsin H and cathepsin B. According to its partially determined amino-acid sequence, human cystatin is highly homologous to egg white cystatin, but only distantly related to stefin, the cytosolic protein inhibitor of cysteine proteinases isolated from human polymorphonuclear granulocytes. Very probably human cystatin is identical with human gamma-trace, a microprotein of known sequence but hitherto unknown function.     

Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin-II.

Oryzacystatin (oryzacystatin-I) is a proteinaceous cysteine proteinase inhibitor (cystatin) in rice seeds and is the first well defined cystatin of plant origin. In this study we isolated cDNA clones for a new type of cystatin (oryzacystatin-II) in rice seeds by screening with the oryzacystatin-I cDNA probe. The newly isolated cDNA clone encodes 107 amino acid residues whose sequence is similar to that of oryzacystatin-I (approximately 55% of identity). These oryzacystatins have no disulfide bonds, and so could be classified as family-I cystatins; however, the amino acid sequences resemble those of family-II members more than family-I members. Oryzacystatin-I and -II are remarkably distinct in two respects: 1) their specificities against cysteine proteinases; and 2) the expression patterns of their mRNAs in the ripening stage of rice seeds. Oryzacystatin-I inhibits papain more effectively (Ki 3.0 x 10(-8) M) than cathepsin H (Ki 0.79 x 10(-6) M), while oryzacystatin-II inhibits cathepsin H (Ki 1.0 x 10(-8) M) better than papain (Ki 0.83 x 10(-6) M). The mRNA for oryzacystatin-I is expressed maximally at 2 weeks after flowering and is not detected in mature seeds, whereas the mRNA for oryzacystatin-II is constantly expressed throughout the maturation stages and is clearly detected in mature seeds. Western blotting analysis using antibody to oryzacystatin-II showed that, as is the case with oryzacystatin-I, oryzacystatin-II occurs in mature rice seeds. Thus, these two oryzacystatin species are believed to be involved in the regulation of proteolysis caused by different proteinases.     

Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases

Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys-to-Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by approximately 20-fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were approximately 14-fold, approximately 10-fold and approximately 300-fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B.     

Expression, purification, and inhibitory activities of mouse cytotoxic T-lymphocyte antigen-2alpha

Cytotoxic T-lymphocyte antigen-2 (CTLA-2) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregion of mouse cathepsin L. Here, we report the expression, purification, and characterization of recombinant CTLA-2 (CTLA-2alpha). CTLA-2alpha was cloned into the pET16b vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) pLysS. The recombinant CTLA-2alpha was highly expressed and purified by His-Bind affinity chromatography, Factor Xa digestion, and hydrophobic chromatography. Throughout these procedures, 3mg recombinant CTLA-2alpha was obtained from 450 ml of bacterial culture medium. The purified protein exhibited inhibitory activities towards certain cysteine proteinases and was properly refolded, as indicated by circular dichroism spectroscopy. Recombinant CTLA-2alpha fully inhibited Bombyx cysteine proteinase (BCP) (overall Kd (Ki*) = 0.23 nM) and and cathepsin L (overall Kd (Ki*) = 0.38 nM). Inhibition of cathepsin H ( Ki = 86 nM) and papain ( Ki = 560 nM) was much weaker, while inhibition of cathepsin B was negligible ( Ki > 1 microM). Our results indicate that mouse CTLA-2alpha is a selective inhibitor of the cathepsin L-like cysteine proteinases.     

Endogenous inhibitor of cysteine proteases from the human kidney

A thermo- and acid stable inhibitor of cysteine proteinases was isolated from the human kidney by successive procedures--acid fractionation, gel-filtration on Sephadex G-75, affinity chromatography on papain-sepharose. The final purification factor was 650 fold. The inhibitor molecular weight was equal to 12 kDa. The values of Ki measured by different methods are (7.9-9.4) X 10(-4) M for papain and (7.1-8.0) X 10(-10) M for purified human kidney cathepsin B. In experiments with papain, inhibitor kass and kd were 1.1 X 10(6) M-1 s-1 and 9.0 X 10(-4) s-1, respectively. The inhibitor did not influence the trypsin activity, its properties being similar to those of related thermo- and acid-stable inhibitors from other human and animal tissues.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-Mr tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sephadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their Mr of about 14 000, their stability with heating at 80 degrees C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (Ki) were determined for these three cysteine proteinases but for cathepsin H, association (kass) and dissociation (kdiss) rate constants were also evaluated. Ki values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, Ki was in the range of 0.1-1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-Mr protein inhibitor in controlling 'in vivo' cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Interaction between chicken cystatin and the cysteine proteinases actinidin, chymopapain A, and ficin

The cysteine proteinase inhibitor cystatin, from chicken egg white, bound with equimolar stoichiometry to the cysteine proteinases actinidin, chymopapain A, and ficin. The changes of near-ultraviolet absorption and fluorescence induced by the binding differed appreciably for the three enzymes, indicating that these spectral changes arise predominantly from aromatic residues in the proteinases. In contrast, the near-ultraviolet circular dichroism changes were similar for all three enzymes, supporting previous evidence that these changes originate mainly from the single tryptophan residue in cystatin, Trp-104. The pseudo-first-order rate constant for the binding increased linearly with the inhibitor concentration up to as high concentrations as could be measured for the three proteinases. This behavior is consistent with the complexes being formed by simple, bimolecular reactions, as was concluded previously for the reaction of cystatin with active and inactivated forms of papain. The second-order association rate constant varied only about 4-fold, from 2.2 X 10(6) to 9.6 X 10(6) M-1.s-1, for the three enzymes, the higher of these values being similar to that measured previously for the reaction with papain. These observations are consistent with the association rate being governed mainly by the frequency of collision between the binding areas of enzyme and inhibitor. All three cystatin-proteinase complexes dissociated to intact inhibitor, demonstrating reversibility. The dissociation rate constants varied about 20000-fold, from 4.6 X 10(-7) s-1 for ficin to 1.1 X 10(-2) s-1 for actinidin, reflecting substantial differences between the enzymes in the nature of the interactions with the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN-III-ompA system

A synthetic gene coding for a chicken egg white cystatin variant was cloned and expressed using the pIN-III-ompA Escherichia coli expression system. After osmotic shock of the E. coli cells, the cysteine proteinase inhibitor was isolated from periplasm and purified by S-carboxymethylpapain affinity chromatography. The resulting inhibitory material was characterized by SDS/PAGE, reversed-phase HPLC, peptide mapping and amino acid sequencing. The recombinant variant chicken AEF-[S1----M, M29----I, M89----L]cystatin shows strong inhibitory activity and displays Ki values in the complex with papain, actinidin and cathepsin B similar to those found for natural chicken cystatin. The purified variant showed a native-chicken-cystatin-like conformational state, as determined by NMR spectroscopy, if the NMR data of 15N-labelled recombinant inhibitor were compared with those of the natural inhibitor.     

The contribution of N-terminal region residues of cystatin A (stefin A) to the affinity and kinetics of inhibition of papain, cathepsin B, and cathepsin L.

The affinity and kinetics of binding of three N-terminally truncated variants of the cysteine proteinase inhibitor cystatin A to cysteine proteinases were characterized. Deletion of Met-1 only minimally altered the inhibitory properties of the protein. However, deletion also of Ile-2 resulted in reduced affinities of 900-, >/=3-, and 200-fold for papain and cathepsins L and B, respectively. Further truncation of Pro-3 substantially increased the inhibition constants to approximately 0.5 microM for papain and cathepsin L and to 60 microM for cathepsin B, reflecting additionally 2 x 10(3)-, 2 x 10(4)-, and 400-fold decreased affinities, respectively. The reductions in affinity shown by the latter mutant indicate that the N-terminal region contributes about 40% of the total free energy of binding of cystatin A to cysteine proteinases. Moreover, Pro-3 and to a lesser extent Ile-2 are the residues responsible for this binding energy. The reduced affinities for papain and cathepsin L were due only to higher dissociation rate constants, whereas both lower association and higher dissociation rate constants contributed to the decreased affinity for cathepsin B. These differential effects indicate that the N-terminal portion of cystatin A primarily functions by stabilizing the complexes with enzymes having easily accessible active-site clefts, e.g., papain and cathepsin L. In contrast, the N-terminal region is required also for an initial binding of cystatin A to cathepsin B, presumably by promoting the displacement of the occluding loop and allowing facile interaction of the rest of the inhibiting wedge with the active-site cleft of the enzyme.     

Expression of Chicken Cystatin for Improving Insect Resistance in Rice

To become mature and infectious, many viruses and insects require proteolytic cleavage, which can be specifically inhibited by proteinase inhibitors. Oryzacystatin (OC), the first-described cystatin originating from rice seed, consists of two molecular species, OC-I and OC-II, both of which have antiviral activity. These intrinsic rice cystatins show a narrow inhibition spectrum and ordinarily are present in rice seeds at insufficient levels for inhibiting the cysteine proteinases of rice insect pests. In addition, our comparison of inhibitory activity (Ki value) showed that chicken cystatin (Ki 5 × 10-12 M) was more powerful than other cystatins, such as OC-I (Ki 3.02 × 10-8 M) and OC-II (l(i 0.83 × 10-8 M). Chicken cystatin also possesses a wide inhibitory spectrum against various cysteine proteinases. Here, we introduced the insecticidal chicken cystatin 8ene into rice plants to improve their insect resistance. Four highly expressive, independent transgenic lines were identified. Molecular analyses revealed that the transferred 8ene was expressed stably in the independent transgenic lines. Therefore, introducing the insecticidal cysteine proteinase inhibitor 8ene into rice plants can be part of a general development strategy for pest control.     

Contributions of individual residues in the N-terminal region of cystatin B (stefin B) to inhibition of cysteine proteinases

The importance of individual residues in the N-terminal region of cystatin B for proteinase inhibition was elucidated by measurements of the affinity and kinetics of binding of N-terminally truncated, recombinant variants of the bovine inhibitor to cysteine proteinases. Removal of Met-1 caused an 8- to 10-fold lower affinity for papain and cathepsin B, decreased the affinity also for cathepsin L but only minimally affected cathepsin H affinity. Additional truncation of Met-2 further weakened the binding to papain and cathepsin B by 40-70-fold, whereas the affinity for cathepsins L and H was essentially unaffected. Removal of Cys-3 had the most drastic effects on the interactions, resulting in a further affinity decrease of approximately 1500-fold for papain, approximately 700-fold for cathepsin L and approximately 15-fold for cathepsin H; the binding to cathepsin B could not be assessed. The binding kinetics could only be evaluated for papain and cathepsin H and showed that the reduced affinities for these enzymes were predominantly due to increased dissociation rate constants. These results demonstrate that the N-terminal region of cystatin B contributes appreciably to proteinase inhibition, in contrast to previous proposals. It is responsible for 12-40% of the total binding energy of the inhibitor to the proteinases investigated, being of least importance for cathepsin H binding. Cys-3 is the most important residue of the N-terminal region for inhibition of papain, cathepsin L and cathepsin H, the role of the other residues of this region varying with the target proteinase.     

Human cathepsin F: expression in baculovirus system, characterization and inhibition by protein inhibitors

Recombinant full-length human procathepsin F, produced in the baculovirus expression system, was partially processed during the purification procedure to a form lacking the N-terminal cystatin-like domain and activated with pepsin. Active cathepsin F efficiently hydrolyzed Z-FR-MCA (kcat/Km=106 mM(-1) s(-1)) and Bz-FVR-MCA (kcat/Km=8 mM(-1) s(-1)), whereas hydrolysis of Z-RR-MCA was very slow (kcat/Km<0.2 mM(-1) s(-1)). Cathepsin F was rapidly and tightly inhibited by cystatin C, chicken cystatin and equistatin with Ki values in the subnanomolar range (0.03-0.47 nM), whereas L-kininogen was a less strong inhibitor of the enzyme (Ki=4.7 nM). Stefin A inhibited cathepsin F slowly (kass=1.6 x 10(5) M(-1) s(-1)) and with a lower affinity (Ki=25 nM). These data suggest that cathepsin F differs from other related endopeptidases by considerably weaker inhibition by stefins.     

The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C.

Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor.     

Arginine-based structures are specific inhibitors of cathepsin C. Application of peptide combinatorial libraries.

Novel synthetic peptide inhibitors of lysosomal cysteine proteinase cathepsin C have been designed through the use of soluble peptide combinatorial libraries. The uncovered structural inhibitory module consists of the N-terminal cluster of L-arginine residues. Its modification with D-amino acids or arginine derivatives did not increase the inhibition strength. Inhibitory potency of oligoarginines improves with the elongation of peptide chain reaching a maximum for octa-L-arginine. The oligoarginines specifically interact with the cathepsin C active site as shown by competitive-type inhibition kinetics (Ki approximately 10-5 M) and intrinsic fluorescence measurements. The inhibitory interaction of oligoarginines is established through the specific spatial contact of a net of guanidino groups in the arginine side-chains, as indicated by comparison with inhibitory action of low molecular mass guanidine derivatives (Ki approximately 10-3 M). Nonarginine polyionic compounds cannot mimic the inhibitory effect of oligoarginines. The arginine-based peptide inhibitors were selective towards cathepsin C among other cysteine proteinases tested.     

Differential effect toward inhibition of papain and cathepsin C by recombinant human salivary cystatin SN and its variants produced by a baculovirus system

Human salivary cystatin SN (CsnSN) is a member of the cystatin superfamily of cysteine proteinase inhibitors. In this study we used a baculovirus expression system to produce a full-length unaltered CsnSN and its variants. The variants were constructed with the changes in the three predicted proteinase-binding regions: the N-terminus (variant N(12-13), G12A-G13A), beta-hairpin loop I (variant L(56-58), Q56G-T57G-V58G) and beta-hairpin loop II (variant L(106-107), P106G-W107G). The secreted CsnSNs were purified using sequential spiral cartridge ultrafiltration and DE-52 radial flow chromatography. The purified proteins were examined for papain- and cathepsin C-inhibition. The wild-type CsnSN, and variants N(12-13) and L(106-107) bound tightly to papain (K(i) < 10 pM), whereas mutation in the loop I reduced binding affinity 5700-fold (K(i) = 57 nM). On the other hand, the wild-type CsnSN bound to cathepsin C less tightly (K(i) = 100 nM). The mutation in the N-terminus or loop I reduced binding affinity by 16 (K(i) = 1.6 microM)- and 19-fold (K(i) = 1.9 microM), respectively, while mutation in loop II resulted in an ineffective cathepsin C inhibitor (K(i) = 14 microM). Collectively, these results suggest that the N-terminal G12-G13 residues of CsnSN are not essential for papain inhibition but play a role in cathepsin C inhibition; residues Q56-T57-V58 in the loop I are essential for both papain and cathepsin C inhibitions, and residues P106-W107 in the loop II are not important for papain inhibition but essential for cathepsin C inhibition. These results demonstrated that CsnSN variants have different effects toward different cysteine proteinases.     

Recombinant chicken egg white cystatin variants of the QLVSG region.

Using recombinant DNA methods, seven cystatin variants were produced by cassette mutagenesis of a chicken egg white cystatin variant which already contains the mutations Ala3, Glu2, Phe1, Ser1-->Met, Met29-->and Met 89-->Leu. When characterized by structural and functional studies, they were all found to harbour mutations in the first hairpin loop, the so-called 'QXVXG' region, which is highly conserved within the cystatin superfamily and thought to be important for its inhibitory activity towards cysteine proteinases. They were purified to more than 90% homogeneity and analysed by SDS/PAGE, HPLC, tryptic peptide mapping, N-terminal amino acid sequencing and ELISA. Structural model building of the variants and their complexes with papain was performed using computer graphics based on the crystallographic coordinates of chicken egg white cystatin and the papain-stefin complex. Only minor conformational changes were required for modelling the mutants or complexes. Equilibrium dissociation constants and rate constants of complex formation of the variants with papain, actinidin as well as cathepsin B and L were determined by kinetic measurements using fluorogenic substrates. The single exchanges Gln53-->Glu, Gln53-->Asn, Val44-->Asp, Gly57-->Ala and the double exchanges Arg52-->Leu, Gln53-->Glu, Gln53-->Asn, Ser56-->Ala, Leu54-->Met, Gly57-->Ala reduced the inhibition of papain, actinidin and cathespin B significantly by 10-1000-fold. With the exception of the Val55-->Asp variant, the differences in the Ki values are mainly due to larger k off values, whereas the kon values seem to be more or less unaffected by the selected mutations. The effect on the inhibition of papain is generally smaller than the effects on actinidin and cathepsin B inhibition. Cathepsin L inhibition is strikingly insensitive to all mutations. These distinct effects of the inhibitor variants indicate differences in proteinase-inhibitor-protein interactions between closely related cysteine proteinases. In addition, the results verify the prediction, made earlier from sequence alignment studies and from a docking model of the chicken cystatin-papain complex, that the first hairpin loop of cystatins is essential for effective inhibition.