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

Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by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.

Download Full-text

Correction to: Stabilized Human Cystatin C, Variant L47C/G69C, is a Better Reporter than the Wild-type Inhibitor for Characterizing the Thermodynamics of Binding to Cysteine Proteases

The Protein Journal ◽

10.1007/s10930-019-09844-5 ◽

2019 ◽

Vol 38 (5) ◽

pp. 608-608

Author(s):

David O. Tovar-Anaya ◽

L. Irais Vera-Robles ◽

M. Teresa Vieyra-Eusebio ◽

Ponciano García-Gutiérrez ◽

Francisco Reyes-Espinosa ◽

...

Keyword(s):

Cystatin C ◽

Cysteine Proteases ◽

Wild Type ◽

Human Cystatin C ◽

Human Cystatin

Download Full-text

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 ◽

10.1002/bip.21140 ◽

2009 ◽

Vol 91 (5) ◽

pp. 373-383 ◽

Cited By ~ 17

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

Download Full-text

Stabilized Human Cystatin C Variant L47C/G69C Is a Better Reporter Than the Wild-Type Inhibitor for Characterizing the Thermodynamics of Binding to Cysteine Proteases

The Protein Journal ◽

10.1007/s10930-019-09839-2 ◽

2019 ◽

Vol 38 (5) ◽

pp. 598-607

Author(s):

David O. Tovar-Anaya ◽

L. Irais Vera-Robles ◽

M. Teresa Vieyra-Eusebio ◽

Ponciano García-Gutiérrez ◽

Francisco Reyes-Espinosa ◽

...

Keyword(s):

Cystatin C ◽

Cysteine Proteases ◽

Wild Type ◽

Human Cystatin C ◽

Human Cystatin

Download Full-text

Interaction of recombinant human cystatin C with the cysteine proteinases papain and actinidin

Biochemical Journal ◽

10.1042/bj2810049 ◽

1992 ◽

Vol 281 (1) ◽

pp. 49-55 ◽

Cited By ~ 73

Author(s):

P Lindahl ◽

M Abrahamson ◽

I Björk

Keyword(s):

Cystatin C ◽

Rate Constants ◽

Proteinase Inhibitors ◽

Equilibrium Constants ◽

Fluorescence Emission ◽

Cysteine Proteinases ◽

Human Cystatin C ◽

Kinetics Of ◽

Human Cystatin ◽

Chicken Cystatin

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.

Download Full-text

Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Journal of Neurochemistry ◽

10.1046/j.1471-4159.2001.00256.x ◽

2001 ◽

Vol 77 (2) ◽

pp. 628-637 ◽

Cited By ~ 26

Author(s):

Miguel Calero ◽

Monika Pawlik ◽

Claudio Soto ◽

Eduardo M. Castaño ◽

Einar M. Sigurdsson ◽

...

Keyword(s):

Cystatin C ◽

Cerebral Hemorrhage ◽

Wild Type ◽

Human Cystatin C ◽

Human Cystatin

Download Full-text

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

Biochemical Journal ◽

10.1042/bj2760387 ◽

1991 ◽

Vol 276 (2) ◽

pp. 387-394 ◽

Cited By ~ 24

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj2730621 ◽

1991 ◽

Vol 273 (3) ◽

pp. 621-626 ◽

Cited By ~ 114

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.

Download Full-text