scholarly journals Human cystatin C. role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leucocyte elastase

1991 ◽  
Vol 273 (3) ◽  
pp. 621-626 ◽  
Author(s):  
M Abrahamson ◽  
R W Mason ◽  
H Hansson ◽  
D J Buttle ◽  
A Grubb ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cystatin C ◽  
Cysteine Proteinase ◽  
Terminal Segment ◽  
Cysteine Proteinase Inhibitor ◽  
Cathepsin H ◽  
Leucocyte Elastase ◽  
Human Cystatin C ◽  
Binding Pockets ◽  
Human Cystatin

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.

scholarly journals Structure and expression of the human cystatin C gene

1990 ◽  
Vol 268 (2) ◽  
pp. 287-294 ◽  
Author(s):  
M Abrahamson ◽  
I Olafsson ◽  
A Palsdottir ◽  
M Ulvsbäck ◽  
Å Lundwall ◽  
...  
Keyword(s):  
Cystatin C ◽  
Proteinase Inhibitor ◽  
Genomic Dna ◽  
Cysteine Proteinase ◽  
Housekeeping Genes ◽  
Genomic Region ◽  
Cysteine Proteinase Inhibitor ◽  
Specific Expression ◽  
Human Cystatin C ◽  
Human Cystatin

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.

The role of the Val57 amino-acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2-L1-beta3 segments of wild-type human cystatin C and its mutants

Biopolymers ◽  
2009 ◽  
Vol 91 (5) ◽  
pp. 373-383 ◽  
Author(s):  
Sylwia Rodziewicz-Motowidło ◽  
Justyna Iwaszkiewicz ◽  
Renata Sosnowska ◽  
Paulina Czaplewska ◽  
Emil Sobolewski ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cystatin C ◽  
Amino Acid Residue ◽  
Wild Type ◽  
Conformational Studies ◽  
Human Cystatin C ◽  
Human Cystatin

Interaction of serum amyloid A with human cystatin C-assessment of amino acid residues crucial for hCC-SAA formation (part II)

2013 ◽  
Vol 26 (9) ◽  
pp. 415-425 ◽  
Author(s):  
Marta Spodzieja ◽  
Monika Rafalik ◽  
Aneta Szymańska ◽  
Aleksandra S. Kołodziejczyk ◽  
Paulina Czaplewska
Keyword(s):  
Amino Acid ◽  
Cystatin C ◽  
Serum Amyloid A ◽  
Serum Amyloid ◽  
Amino Acid Residues ◽  
Amyloid A ◽  
Human Cystatin C ◽  
Human Cystatin

scholarly journals Amino acid substitutions in the N-terminal segment of cystatin C create selective protein inhibitors of lysosomal cysteine proteinases

1998 ◽  
Vol 330 (2) ◽  
pp. 833-838 ◽  
Author(s):  
W. Robert MASON ◽  
Katia SOL-CHURCH ◽  
Magnus ABRAHAMSON
Keyword(s):  
Amino Acid ◽  
Cystatin C ◽  
Cathepsin L ◽  
Specific Inhibitor ◽  
Terminal Segment ◽  
Cathepsin S ◽  
Amino Acid Substitutions ◽  
Cysteine Proteinases ◽  
Aromatic Residues ◽  
Human Cystatin C

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.

scholarly journals Importance of the evolutionarily conserved glycine residue in the N-terminal region of human cystatin C (Gly-11) for cysteine endopeptidase inhibition

1993 ◽  
Vol 291 (1) ◽  
pp. 123-129 ◽  
Author(s):  
A Hall ◽  
H Dalbøge ◽  
A Grubb ◽  
M Abrahamson
Keyword(s):  
Cystatin C ◽  
Equilibrium Constants ◽  
Terminal Segment ◽  
Site Directed Mutagenesis ◽  
Side Chains ◽  
Wild Type ◽  
Human Cystatin C ◽  
Evolutionarily Conserved ◽  
Cysteine Endopeptidases ◽  
Human Cystatin

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.

scholarly journals Papain labelled with fluorescent thiol-specific reagents as a probe for characterization of interactions between cysteine proteinases and their protein inhibitors by competitive titrations

1991 ◽  
Vol 276 (2) ◽  
pp. 387-394 ◽  
Author(s):  
P Lindahl ◽  
E Raub-Segall ◽  
S T Olson ◽  
I Björk
Keyword(s):  
Cystatin C ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Equilibrium Constants ◽  
Sulphonic Acid ◽  
Cysteine Proteinases ◽  
Human Cystatin C ◽  
Human Cystatin ◽  
Chicken Cystatin

Papain was labelled by attachment of the fluorescent groups 2-(4′-acetamidoanilino)naphthalene-6-sulphonic acid (AANS) or N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid (AEDANS) to the active-site cysteine residue, with the aim of using the labelled papains as probes in competitive titrations of unlabelled cysteine proteinases with their inhibitors. The interaction between the labelled papains and cystatins was accompanied by an increase in fluorescence emission of up to 38-fold for AANS-papain and approximately 3.5-fold for AEDANS-papain. Fluorescence titrations gave dissociation equilibrium constants of 3.1 and 0.6 microM for the binding of chicken cystatin and recombinant human cystatin C respectively to AANS-papain and of 11.9 microM for the binding of chicken cystatin to AEDANS-papain. The kinetics of interaction of chicken cystatin with AANS-papain showed an unusual biphasic dependence of the observed pseudo-first-order rate constant on inhibitor concentration, consistent with the reaction occurring via both pathways of a general two-step binding mechanism. AANS-papain was selected as the most suitable probe for competitive titrations of unlabelled active or inactivated cysteine proteinases with inhibitors. This technique, which provides stoichiometries and dissociation constants for the interaction between unlabelled enzyme and inhibitor, allows monitoring of the interactions by a large fluorescent signal in a wavelength region where the interacting proteins do not contribute to the observed fluorescence. Such competitive titrations of active papain or actinidin with chicken cystatin or recombinant human cystatin C all gave inhibitor/enzyme stoichiometries of close to 1.0. A dissociation constant of 1.8 microM for the reaction of chicken cystatin with a papain derivative, S-[N-(3-carboxypropyl)succinimidyl]-papain, was also determined by the same technique. These results show the usefulness of the fluorescent papains for the characterization of interactions between cysteine-proteinase inhibitors and their target enzymes.

scholarly journals Purification and characterization of a new cystatin inhibitor from Taiwan cobra (Naja naja atra) venom

1998 ◽  
Vol 331 (1) ◽  
pp. 239-244 ◽  
Author(s):  
Michèle BRILLARD-BOURDET ◽  
Vinh NGUYÊN ◽  
Michèle FERRER-DI MARTINO ◽  
Francis GAUTHIER ◽  
Thierry MOREAU
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Cysteine Proteinase Inhibitor ◽  
Naja Naja ◽  
Cystatin M ◽  
Human Cystatin ◽  
Naja Atra

Cobra cystatin, a new cysteine-proteinase inhibitor of the cystatin superfamily, was isolated from the venom of the Taiwan cobra (Naja naja atra) by affinity chromatography on S-carboxymethylpapain–Sepharose and reverse-phase chromatography. The venom contained two forms of the inhibitor, one of 11870 Da and the other of 12095 Da, as determined by MS, and pI values of 6.2 and 6.1. Cobra cystatin strongly inhibits cysteine proteinases of the papain family, but not calpain. Papain, cathepsin L, cathepsin B and cathepsin S are inhibited with Ki values of 0.19, 0.1, 2.5 and 1.2 nM respectively. The amino acid sequence of cobra cystatin shows that it is a Type 2 cystatin. The amino acid sequence is 73% identical with that of the cystatin in African-puff-adder (Bitis arietans) venom, with which it shares a unique six-residue insertion in a region opposite the reactive inhibitory site. Cobra cystatin is 25–42% identical with other Type 2 cystatins, the most closely related being the recently described human cystatin M, which also has a similar five-residue insertion starting at position 76 (chicken cystatin numbering). A molecular phylogenetic tree of 16 representative members of Family 2 cystatins was constructed by parsimony analysis; it suggests that snake cystatins, together with Tachypleus tridentatus (Japanese horseshoe crab) cystatin and human cystatin M, form a new subfamily within cystatin Family 2.

scholarly journals NMR and crystallographic structural studies of the extremely stable monomeric variant of human cystatin C with single amino acid substitution

FEBS Journal ◽  
2019 ◽  
Vol 287 (2) ◽  
pp. 361-376 ◽  
Author(s):  
Martyna Maszota‐Zieleniak ◽  
Przemyslaw Jurczak ◽  
Marta Orlikowska ◽  
Igor Zhukov ◽  
Dominika Borek ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cystatin C ◽  
Amino Acid Substitution ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Structural Studies ◽  
Human Cystatin C ◽  
Human Cystatin
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