Structural Basis for the Biological Specificity of Cystatin C

Abstract

The structural basis for the biological specificity of human cystatin C has been investigated. Cystatin C and other inhibitors belonging to family 2 of the cystatin superfamily interact reversibly with target peptidases, seemingly by independent affinity contributions from a wedge-shaped binding region built from two loop-forming inhibitor segments and a binding region corresponding to the N-terminal segment of the inhibitor. Human cystatin C variants with Gly substitutions for residues Arg-8, Leu-9, and/or Val-10 of the N-terminal binding region, and/or the evolutionarily conserved Trp-106 in the wedge-shaped binding region, were produced by site-directed mutagenesis and Escherichia coli expression. A total of 10 variants were isolated, structurally verified, and compared to wild-type cystatin C with respect to inhibition of the mammalian cysteine peptidases, cathepsins B, H, L, and S. Varying contributions from the N-terminal binding region and the wedge-shaped binding region to cystatin C affinity for the four target peptidases were observed. Interactions from the side chains of residues in the N-terminal binding region and Trp-106 are jointly responsible for the major part of cystatin C affinity for cathepsin L and are also of considerable importance for cathepsin B and H affinity. In contrast, for cathepsin S inhibition these interactions are of lesser significance, as reflected by a Ki value of 10(-8) M for the cystatin C variant devoid of Arg-8, Leu-9, Val-10, and Trp-106 side chains. The side chain of Val-10 is responsible for most of the affinity contribution from the N-terminal binding region, for all four enzymes. The contribution of the Arg-8 side chain is minor, but significant for cystatin C interaction with cathepsin B. The Leu-9 side chain confers selectivity to the inhibition of the target peptidases; it contributes to cathepsin B and L affinity by factors of 200 and 50, respectively, to cathepsin S binding by a factor of 5 only, and results in a 10-fold decreased affinity between cystatin C and cathepsin H.

Cystatin C is a new marker for left ventricular hypertrophy in patients with chronic kidney disease

“…Это обусловлено тем, что цистатин С, синтезируемый всеми ядерными клетками, свободно фильтруется через клубочковую мембрану и полностью метаболизируется после его реабсорбции в проксималь-ных канальцах и в настоящее время, по данным литерату-ры [11], является показателем функции почек.…”

Cystatin F Is a Glycosylated Human Low Molecular Weight Cysteine Proteinase Inhibitor

“…This contrasts with the inhibitory specificities of cystatins C and E/M, which both have the capacity to inhibit cathepsin B (Table I). A structural element that has been shown to be at least partly responsible for the target enzyme specificity of cystatin C and of crucial importance for efficient cystatin C binding of cathepsin B is the N-terminal segment with ArgLeu-Val-Gly at positions 8 -11 (40,53). Leu-9 and Val-10, interacting with target enzyme substrate subpockets S 3 and S 2 , respectively, are especially important for a fast interaction between the cystatin and cathepsin B (54,55).…”

“…Enzyme Assays-The methods used for active-site titration of papain (using benzoyl-DL-Arg p-nitroanilide (Bachem Feinchemikalien, Bubendorf, Switzerland) as substrate), titration of the molar enzyme inhibitory concentration in cystatin F preparations, and determination of equilibrium constants for dissociation (K i ) of complexes between cystatin F and cysteine proteinases have been reviewed (1). The enzymes used were papain (EC 3.4.22.2; from Sigma), activatable to 70 -75% after affinity purification on Sepharose-coupled Gly-Gly-Tyr-Arg as detailed previously (39,40), human cathepsin B (EC 3.4.22.1; from Calbiochem, La Jolla, CA), and human cathepsin L (EC 3.4.22.15; Calbiochem). The fluorogenic substrate used for K i determinations was benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin (10 M; Bachem Feinchemikalien), and the assay buffer was 100 mM sodium phosphate buffer (adjusted to pH 6.5 for papain and to pH 6.0 for cathepsin B and cathepsin L) containing 1 mM dithiothreitol and 2 mM EDTA.…”

“…For fractionation of blood plasma, solid ammonium sulfate was added to a series of tubes containing 2-ml portions of plasma to achieve 25,30,40,45,50,55,60,70, and 75% (w/v) saturation. Following overnight incubation at 4°C, the tubes were centrifuged (10,000 ϫ g, 30 min), the supernatants were collected, and the precipitates were dissolved in water.…”

Application of polymannosylated cystatin to surimi from roe‐herring to prevent gel weakening

“…Heating the polymannosylated protein at 95³C for 30 min resulted in less than 40% reduction of the papain-inhibiting activity. Although it is reported that cystatin C is resistant to heating due to the tightly packed conformation [15], further enhancement of the thermostability was observed in the polymannosylated cystatin.…”