| 1. |
- Abrahamson, Magnus, et al.
(författare)
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Human cystatin C: Role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leucocyte elastase
- 1991
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Ingår i: Biochemical Journal. - 0264-6021. ; 273:3, s. 621-626
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Tidskriftsartikel (refereegranskat)abstract
- Leucocyte elastase in catalytic amounts was observed to rapidly cleave the Val-10-Gly-11 bond of the human cysteine-proteinase inhibitor cystatin C at neutral pH. The resulting modified inhibitor had size and amino acid composition consistent with a cystatin C molecule devoid of the N-terminal Ser-1-Val-10 decapeptide. Leucocyte-elastase-modified cystatin C had more than 240-fold lower affinity than native cystatin C for papain. Removal of the N-terminal decapeptide of human cystatin C also decreased inhibition of human cathepsins B and L by three orders of magnitude, but decreased inhibition of cathepsin H by only 5-fold. A tripeptidyldiazomethane analogue of of the N-terminal portion of cystatin C was a good inhibitor of cathepsins B and L but a poor inhibitor of cathepsin H. It therefore appears that amino acid side chains of the N-terminal segment of cystatin C bind in the substrate-binding pockets of cathepsins B and L but not in those of cathepsin H. It is argued that the N-terminal cystatin C interaction with cathepsin B is physiologically important and hence that leucocyte elastase could have a function as a regulator of extracellular cysteine-proteinase inhibitory activity at sites of inflammation.
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| 2. |
- Abrahamson, Magnus, et al.
(författare)
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Structure and expression of the human cystatin C gene
- 1990
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Ingår i: Biochemical Journal. - 1470-8728. ; 268:2, s. 287-294
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Tidskriftsartikel (refereegranskat)abstract
- The structural organization of the gene for the human cysteine-proteinase inhibitor cystatin C was studied. Restriction-endonuclease digests of human genomic DNA hybridized with human cystatin C cDNA and genomic probes produced patterns consistent with a single cystatin C gene and, also, the presence of six closely related sequences in the human genome. A 30 kb restriction map covering the genomic region of the cystatin C gene was constructed. The positions of three polymorphic restriction sites, found at examination of digests of genomic DNA from 79 subjects, were localized in the flanking regions of the gene. The gene was cloned and the nucleotide sequence of a 7.3 kb genomic segment was determined, containing the three exons of the cystatin C structural gene as well as 1.0 kb of 5'-flanking and 2.0 kb of 3'-flanking sequences. Northern-blot experiments revealed that the cystatin C gene is expressed in every human tissue examined, including kidney, liver, pancreas, intestine, stomach, antrum, lung and placenta. The highest cystatin C expression was seen in seminal vesicles. The apparently non-tissue-specific expression of this cysteine-proteinase inhibitor gene is discussed with respect to the structure of its 5'-flanking region, which shares several features with those of housekeeping genes.
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| 3. |
- Bjork, I, et al.
(författare)
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Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes
- 1994
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Ingår i: Biochemical Journal. - 0264-6021. ; 299, s. 219-225
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Tidskriftsartikel (refereegranskat)abstract
- The importance of the N-terminal region of human cystatin C or chicken cystatin for the kinetics of interactions of the inhibitors with four cysteine proteinases was characterized. The association rate constants for the binding of recombinant human cystatin C to papain, ficin, actinidin and recombinant rat cathepsin B were 1.1 x 10(7), 7.0 x 10(6), 2.4 x 10(6) and 1.4 x 10(6) M-1.s-1, whereas the corresponding dissociation rate constants were 1.3 x 10(-7), 9.2 x 10(-6), 4.6 x 10(-2) and 3.5 x 10(-4) s-1. N-Terminal truncation of the first ten residues of the inhibitor negligibly affected the association rate constant with papain or ficin, but increased the dissociation rate constant approx. 3 x 10(4)- to 2 x 10(6)-fold. In contrast, such truncation decreased the association rate constant with cathepsin B approx. 60-fold, while minimally affecting the dissociation rate constant. With actinidin, the truncated cystatin C had both an approx. 15-fold lower association rate constant and an approx. 15-fold higher dissociation rate constant than the intact inhibitor. Similar results were obtained for intact and N-terminally truncated chicken cystatin. The decreased affinity of human cystatin C or chicken cystatin for cysteine proteinases after removal of the N-terminal region is thus due to either a decreased association rate constant or an increased dissociation rate constant, or both, depending on the enzyme. This behaviour indicates that the contribution of the N-terminal segment of the two inhibitors to the interaction mechanism varies with the target proteinase as a result of structural differences in the active-site region of the enzyme.
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| 4. |
- Björk, Ingemar, et al.
(författare)
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Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases
- 1995
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Ingår i: Biochemical Journal. - 0264-6021. ; 306:2, s. 513-518
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between cystatin C variants, in which the evolutionarily conserved Gly-11 residue was substituted by Ala, Glu or Trp, and the cysteine proteinases, papain, ficin, actinidin and cathepsin B, was characterized. The substitutions reduced the affinity of binding in a manner consistent with the Gly residue of the wild-type inhibitor, allowing the N-terminal region to adopt a conformation that was optimal for interaction with target proteinases. Replacement of Gly-11 by Ala resulted in only a 5- to 100-fold reduction in binding affinity. Comparison with the affinities of wild-type cystatin C lacking the N-terminal region indicated that even this small structural change affects the conformation of this region sufficiently to largely abolish its interaction with the weakly binding proteinases, actinidin and cathepsin B. However, the substitution allows interactions of appreciable strength between the N-terminal region and the tightly binding enzymes, papain or ficin. Replacement of Gly-11 with the larger Glu and Trp residues substantially decreased the affinity of binding to all enzymes, from 10(3)- to 10(5)-fold. These substitutions further affect the conformation of the N-terminal region, so that interactions of this region with papain and ficin are also essentially eliminated. The decreased affinities of the three cystatin C variants for papain, ficin and actinidin were due exclusively to increased dissociation rate constants. In contrast, the decreased affinity between cathepsin B and the Ala-11 variant, the only one for which rate constants could be determined with this enzyme, was due almost entirely to a decreased association rate constant. This behaviour is analogous to that observed for forms of cystatin C lacking the N-terminal region and supports the conclusion that the mode of interaction of this region with target proteinases varies with the enzyme as a result of structural differences in the active-site region of the latter.
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| 5. |
- Buttle, David J, et al.
(författare)
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Human sputum cathepsin B degrades proteoglycan, is inhibited by a2-macroglobulin and modulated by neutrophil elastase cleavage of cathepsin B precursor and cystatin C
- 1991
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Ingår i: Biochemical Journal. - 0264-6021. ; 276, s. 325-331
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Tidskriftsartikel (refereegranskat)abstract
- The high-Mr alkali-stable form of cathepsin B was purified from purulent human sputum. It was shown to solubilize proteoglycan monomer entrapped in polyacrylamide at a rate comparable with that of human lysosomal cathepsin B. Like the enzyme from lysosomes, sputum cathepsin B was bound by human alpha 2-macroglobulin, which inhibited its action on proteoglycan. Cystatin C in purulent sputum was shown to be the N-terminally truncated form generated by neutrophil elastase cleavage, and sputum cathepsin B was only weakly inhibited by recombinant cystatin C that had been cleaved by neutrophil elastase in vitro. Addition of neutrophil elastase to mucoid sputum led to a 5-fold increase in cathepsin B activity concomitant with a lowering in Mr of the cysteine proteinase from 40,000 to 37,000, i.e. the size of the active enzyme purified from purulent sputum. It is concluded that the high-Mr form of cathepsin B present in purulent sputum is a functional proteinase, unlike similar forms of the enzyme secreted by mammary gland in organ culture. The activity of cathepsin B in sputum is modulated by neutrophil elastase, by a combination of inhibitor inactivation and zymogen activation.
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| 6. |
- Hall, Anders, et al.
(författare)
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Importance of the evolutionarily conserved glycine residue in the N-terminal region of cystatin C (Gly-11) for cysteine endopeptidase inhibition
- 1993
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Ingår i: Biochemical Journal. - 0264-6021. ; 291:1, s. 123-129
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Tidskriftsartikel (refereegranskat)abstract
- Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by site-directed mutagenesis and expression in Escherichia coli. The five variants were isolated and structurally verified. Their inhibitory properties were compared with those of wild-type recombinant cystatin C by determination of the equilibrium constants for dissociation (Ki) of their complexes with the cysteine endopeptidases papain and human cathepsin B and with the cysteine exopeptidase dipeptidyl peptidase I. The Ser-11 and Ala-11 cystatin C variants displayed Ki values for the two endopeptidases that were approx. 20-fold higher than those of wild-type cystatin C, while the corresponding values for the Trp-11. Arg-11 and Glu-11 variants were increased by a factor of about 2000. In contrast, the Ki values for the interactions of all five variants with the exopeptidase differed from that of wild-type cystatin C by a factor of less than 10. Wild-type cystatin C and the Ser-11, Ala-11 and Glu-11 variants were incubated with neutrophil elastase, which in all cases resulted in the rapid hydrolysis of a single peptide bond, between amino acid residues 10 and 11. The Ki values for the interactions with papain of these three N-terminal-decapeptide-lacking cystatin C variants were 20-50 nM, just one order of magnitude higher than the value for N-terminally truncated wild-type cystatin C, which in turn was similar to the corresponding values for the full-length Glu-11, Arg-11 and Trp-11 variants. These data indicate that the crucial feature of the conserved Gly residue in position 11 of wild-type cystatin C is that this residue, devoid of a side-chain, will allow the N-terminal segment of cystatin C to adopt a conformation suitable for interaction with the substrate-binding pockets of cysteine endopeptidases, resulting in high-affinity binding and efficient inhibition. The functional properties of the remaining part of the proteinase contact area, which is built from more C-terminal inhibitor segments, are not significantly affected even when amino acids with bulky or charged side-chains replace the Gly-11 residue of the N-terminal segment.
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| 7. |
- Lindahl, P, et al.
(författare)
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Interaction of recombinant human cystatin C with the cysteine proteinases papain and actinidin
- 1992
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Ingår i: Biochemical Journal. - 0264-6021. ; 281:1, s. 49-55
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between recombinant human cystatin C and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The absorption, near-u.v.c.d. and fluorescence-emission difference spectra for the cystatin C-proteinase interactions were all found to be similar to the corresponding spectra for chicken cystatin. The kinetics of binding of cystatin C to the two enzymes were best described by a simple reversible one-step bimolecular mechanism, like the kinetics of the reaction of chicken cystatin with several cysteine proteinases. Moreover, the second-order association rate constants at 25 degrees C, pH 7.4 and I0.15, of 1.1 x 10(7) and 2.4 x 10(6) M-1.s-1 for the reactions of cystatin C with papain and actinidin respectively, were similar to the corresponding rate constants for the chicken inhibitor and close to the value expected for a diffusion-controlled rate. The dissociation equilibrium constants, approx. 11 fM and approx. 19 nM for the binding of cystatin C to papain and actinidin respectively, were also comparable with the dissociation constants for chicken cystatin. The affinity between cystatin C and several inactivated papains or actinidins decreased with increasing size of the inactivating group in a manner similar to that in earlier studies with the chicken inhibitor. Together, these results strongly indicate that the mechanisms of the reactions of cystatin C and chicken cystatin with cysteine proteinases are identical or highly similar, but differ from that of reactions between serine-proteinase inhibitors and their target enzymes. The model for the proteinase-inhibitor interaction, based on the X-ray structure of chicken cystatin, therefore should be largely applicable also to human cystatin C.
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| 8. |
- Mason, R W, et al.
(författare)
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Amino acid substitutions in the N-terminal segment of cystatin C create selective inhibitors of lysosomal cysteine proteinases
- 1998
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Ingår i: Biochemical Journal. - 0264-6021. ; 330:2, s. 833-838
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Tidskriftsartikel (refereegranskat)abstract
- We used site-directed mutagenesis to alter the specificity of human cystatin C, an inhibitor with a broad reactivity against cysteine proteinases. Nine cystatin C variants containing amino acid substitutions in the N-terminal (L9W, V10W, V10F and V10R) and/or the C-terminal (W106G) enzyme-binding regions were designed and produced in Escherichia coli. It was discovered that the inhibition profile of the cystatin could be altered by changing residues 9 and 10, which are proposed to bind in the S3 and S2 substrate-binding pockets respectively of the enzymes. All of the variants with substitutions in the N-terminal segment displayed decreased binding to cathepsins B and H, indicating that the S3 and S2 pockets of these enzymes cannot easily accommodate large aromatic residues. The introduction of a charged residue into S2 (variant V10R) created a more specific inhibitor to distinguish cathepsin B from cathepsin H. Cathepsin L showed a preference for larger aromatic residues in S2. In contrast, cathepsin S preferred phenylalanine to valine in S2, but bound less tightly to the V10W cystatin variant. The latter variant proved to be valuable for discriminating between cathepsin L and cathepsin S (Ki 2.4 and 190 pM respectively). The equilibrium dissociation constant of the complex between cathepsin L and variant L9W/W106G showed little difference in affinity from that of the cathepsin L complex with the singly substituted W106G variant. In contrast, the L9W/W106G variant displayed increased specificity for cathepsin S with a Ki of 10 pM. Our results clearly indicate differences in the specificity of interaction between the N-terminal region of cystatin C and cathepsins B, H, L and S, and that, although cystatin C has evolved to be a good inhibitor of all of the mammalian cysteine proteinases, more specific inhibitors of the individual enzymes can be engineered.
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| 9. |
- Salvesen, Guy, et al.
(författare)
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Human low-Mr kininogen contains three copies of a cystatin sequence that are divergent in structure and in inhibitory activity for cysteine proteinases.
- 1986
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Ingår i: Biochemical Journal. - 0264-6021. ; 234:2, s. 429-434
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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