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PubMed2003

Evaluation of an IgG cystatin capture enzyme-linked immunosorbent assay for the detection of anti-cysteine proteinase antibodies in asymptomatic trichomoniasis patients.

All isolates of T. vaginalis release cysteine proteinases proteolytic enzymes that are shed into the vagina or culture medium. Cystatin has been used successfully as a capture agent in ELISA to detect cysteine proteinase antibodies without the need for purified proteinases. The ELISA was evaluated in comparison to wet mount microscopy and culture techniques. IgG cystatin capture ELISA proved to be a sensitive and highly specific (100%) assay that could rapidly detect anti-cysteine proteinase antibodies in both vaginal washouts and sera of asymptomatic patients with a sensitivity of 100% and 86.7%, respectively. A defined discrimination between sero-positive and sero-negative individuals was markedly observed for ELISA-vaginal washouts providing a more conclusive diagnosis by this technique. The results demonstrated that in trichomoniasis patients (52 cases) whether symptomatic or asymptomatic. T. vaginalis infection was detected in 19 (36.5%), 49 (94.2%), 50 (96.1%) and 48 (92.3%) by wet mount, culture, cystatin capture ELISA-vaginal washouts and ELISA-sera, respectively. The assay was reliable also as a test of cure with a specificity of 94.4% in the vaginal washouts and 83.3% in sera.

Adult↗
PubMed2003

[Cystatin C in the evaluation of renal function].

Glomerular filtration rate (GFR) is the best indicator of renal function. GFR is usually estimated by serum creatinine or the creatinine clearance calculated on urine collected over 24 hours or with the Cockcroft formula. These methods are however limited. Serum creatinine has a very poor sensitivity and urine collection is difficult. Cystatin C is a protease inhibitor produced in a constant manner by nucleated cells. This molecule is freely filtrated by the glomerule and quite completely catabolized in the proximal tubules. Its plasmatic concentration might thus be used to estimate GFR. Presently available data allow to conclude that plasmatic cystatin C is at least as good as serum creatinine to estimate GFR. It is less sensible to changes in body mass. Its determination appears more sensitive to detect early mild changes in GFR. Reference values are presently available for the different methods of determination. Cystatin C plasma level determination is more expensive than routine creatinine plasma determination. In the absence of very significant advantages, this might explain its limited use in daily clinical practice.

Biomarkers↗
PubMed2005

Late stage inhibition of hematogenous melanoma metastasis by cystatin C over-expression.

BACKGROUND: Tumor metastasis is a frequent cause of treatment failure for cancer patients. A key feature of metastatic cancer cells is their invasive ability. Cysteine proteases contribute to invasive properties of many cancer cell types. To analyze the contribution of cysteine proteases to metastasis we have over-expressed in B16 melanoma cells the natural cysteine protease inhibitor, cystatin C. We measured in vitro invasion of cystatin over-expression clones with Boyden chamber type assays. Tail-vein injections of cells were used to compare lung tumor colonization. Subcutaneous tumor growth and tumor cell metastasis from primary tumors were also analyzed. Apoptosis of tumor cells was measured in lung tissues following melanoma cell injection. RESULTS: Results show the in vitro invasion of cystatin C over-expressing cells was dramatically inhibited. Lung tumor colonization was also reduced. Increased tumor cell apoptosis was found to be an important factor and may be related to the reduced tumor burden noted in this system of melanoma metastasis. CONCLUSION: Cysteine proteases therefore, may be a target for future anti-metastatic therapies.

Journal Article↗
PubMed1989

The arrangement of disulfide loops in human alpha 2-HS glycoprotein. Similarity to the disulfide bridge structures of cystatins and kininogens.

The complete disulfide loop structure of human alpha 2-HS glycoprotein has been elucidated. alpha 2-HS glycoprotein isolated from human plasma was found to be a two-chain protein composed of a heavy and a light chain. The heavy chain comprises the A-chain of alpha 2-HS glycoprotein (Yoshioka, Y., Gejyo, F., Marti, T., Rickli, E. E., Bürgi, W., Offner, G. D., Troxler, R. F., and Schmid, K. (1986) J. Biol. Chem. 261, 1665-1676) and part of the connecting peptide which has been predicted from the corresponding cDNA sequence (Lee, C. C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407), whereas the light chain corresponds to the beta-chain of alpha 2-HS glycoprotein (Gejyo, F., Chang, J. L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R. F., Van Halbeek, H., Dorland, L., Gerwig, G. J., Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258, 4966-4971). Twelve half-cystine residues are present in the alpha 2-HS glycoprotein molecule, and 11 of them are positioned in the heavy chain and a single one in the light chain of the molecule; they form six disulfide bridges. The first and the last half-cystine residues of the amino acid sequence of alpha 2-HS glycoprotein are engaged in the formation of a loop spanning the extreme NH2- and COOH-terminal portions of the molecule, thereby connecting the heavy and light chains. The other 10 half-cystines residues are linked consecutively in the heavy chain and form five loops which span 4-19 amino acid residues. Among them are two pairs of loops which are characterized by mutual sequence homology. The particular arrangement of disulfide loops in alpha 2-HS glycoprotein is similar to the patterns of linearly arranged and tandemly repeated disulfide loops of cysteine proteinase inhibitors, i.e. the cystatins and the kininogens. It is concluded that alpha 2-HS glycoprotein represents a structural prototype of a novel family among the cystatin superfamily, characterized by the presence of two cystatin-like building blocks. Extensive similarity among the NH2-terminal sequences of alpha 2-HS glycoprotein and human histidine-rich glycoprotein suggest that the latter protein is another candidate protein of this new family.

Amino Acid Sequence↗
PubMed1999

Modulation of invasive properties of murine squamous carcinoma cells by heterologous expression of cathepsin B and cystatin C.

Murine SCC-VII squamous carcinoma cells have the capacity to penetrate reconstituted basement membranes (Matrigel) in vitro. The invasion of Matrigel layers by SCC-VII cells was significantly reduced by E-64, a specific inhibitor of lysosomal cysteine proteinases. The cathepsin-B-selective E-64 derivative, CA-074, inhibited penetration of Matrigel by SCC-VII cells to the same extent, indicating a major role for this particular lysosomal enzyme in extracellular-matrix degradation during squamous-carcinoma-cell invasion. SCC-VII cells were stably transfected with a cDNA encoding human procathepsin B, in an attempt to modulate the invasive properties of the cell line. The transfected cells expressed the heterologous gene, secreted increased amounts of procathepsin B and displayed enhanced invasive potential. In vivo, the activity of cathepsin B is strictly regulated by endogenous inhibitors. SCC-VII cells were therefore also stably transfected with a cDNA encoding human cystatin C, the most potent cysteine-proteinase inhibitor in mammalian tissues. The expression of this transgene resulted in the production of active recombinant cystatin C and a pronounced reduction in Matrigel invasion. These studies demonstrate that the invasive properties of squamous-cell carcinomas can be changed by modulation of the balance between cathepsin B and its endogenous inhibitors, and provide further evidence for the involvement of this lysosomal cysteine proteinase in tumour invasion and metastasis.

Animals↗
PubMed2005

Truncated cystatin C in cerebrospiral fluid: Technical [corrected] artefact or biological process?

Cystatin C, a low molecular weight cysteine proteinase inhibitor present in human body fluids at physiological concentrations, is more expressed in cerebrospinal fluid (CSF) than in plasma. Mass spectrometric characterization showed that after 3 months of storage of human CSF at -20 degrees C, cystatin C was cleaved in the peptide bond between R8 and L9 and lost its eight N-termini amino acids, whereas this cleavage did not occur when stored at -80 degrees C. This truncation occurred in all CSF samples studied irrespective of the underlying neurological status, indicating a storage-related artefact rather than a physiological or pathological processing of the protein. These results stress the importance of optimal preanalytical storage conditions of any sample prior to proteomics studies.

Artifacts↗
PubMed2000

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.

Animals↗
PubMed1997

The NNP-1 gene (D21S2056E), which encodes a novel nuclear protein, maps in close proximity to the cystatin B gene within the EPM1 and APECED critical region on 21q22.3.

We have cloned and molecularly characterized a novel human cDNA that encodes a protein of about 52 kDa that was shown by immunocytochemistry to have a nuclear localization. Northern blot analysis of a variety of human tissues revealed that the gene for this novel nuclear protein, designated NNP-1 (HGMW-approved symbol D21S2056E), is ubiquitously expressed as a 2.0-kb transcript. The NNP-1 protein displays significant sequence similarity to the C47E12.7 protein of Caenorhabditis elegans (38% identity and 58% similarity) and the YD78 protein of Saccharomyces cerevisiae (36% identity and 54% similarity). The human NNP-1 gene was mapped to a 15-kb DNA interval on chromosome 21q22.3, close to markers D21S1459 and D21S1953, that is 25 kb distal to the gene encoding cystatin B. Finally, an as-yet unknown gene that is highly related to NNP-1 was found proximal to the gene encoding cystatin B, near marker D21S1458, which is approximately 75 kb centromeric to the NNP-1 gene.

Amino Acid Sequence↗
PubMed2005

Influence of commonly used drugs on the accuracy of cystatin C-derived glomerular filtration rate.

There is controversy about the effect of certain drugs on cystatin C (CysC) concentrations, which would limit the usability of CysC for estimation of glomerular filtration rate (GFR) in patients with renal disease. Seventy-one children (ages 2.6 months to 18 years) with renal disease and on at least one study medication (tacrolimus, cyclosporine, mycophenolate mofetil, corticosteroids, fosinopril, ramipril and enalapril, losartan, cotrimoxazole) were tested in 85 nuclear medicine GFR clearance studies with simultaneous CysC determinations. We analyzed the relationship between the dose per kilogram and the ratio of the measured GFR to the CysC-derived GFR, with a ratio of 1 resembling agreement. A non-zero slope in linear regression analysis was considered significant for a drug effect on CysC. No significant relationship was found between the doses of the medication and the cystatin C GFR for any of the medications. Only cotrimoxazole showed a GFR ratio that was significantly lower than 1, which may be related to small numbers; otherwise the value was always 1. CysC provides accurate data for calculating GFR independent of the drug doses studied and avoids the use of methods of direct GFR measurement.

Adolescent↗
PubMed1989

Immunochemical quantitation of cysteine proteinase inhibitor cystatin C in inflamed human gingiva.

The amount of the low molecular-weight inhibitor, cystatin C, was determined by the enzyme-linked immunosorbent assay. Gingival tissue samples were obtained during periodontal surgery from 22 patients with different degrees of inflammatory periodontal disease, as indicated by gingival index and probing depth (PD). The concentration of cystatin C was in the range from 0.21 to 3.82 micrograms/g tissue and was significantly decreased (p less than 0.01) in samples taken from sites with increased PD.

Adult↗
PubMed1991

The cystatins: protein inhibitors of cysteine proteinases.

The last decade has witnessed enormous progress of protein inhibitors of cysteine proteinases concerning their structures, functions and evolutionary relationships. Although they differ in their molecular properties and biological distribution, they are structurally related proteins. All three inhibitory families, the stefins, the cystatins and the kininogens, are members of the same superfamily. Recently determined crystal structures of chicken cystatin and human stefin B established a new mechanism of interaction between cysteine proteinases and their inhibitors which is fundamentally different from the standard mechanism for serine proteinases and their inhibitors.

Amino Acid Sequence↗
PubMed1995

Inhibition of cruzipain, the major cysteine proteinase of the protozoan parasite, Trypanosoma cruzi, by proteinase inhibitors of the cystatin superfamily.

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi epimastigotes, purified to a sequentially pure form, exists in multiple forms with pI values between 3.7 and 5.1, and an apparent molecular mass of 41 kDa. The enzyme is stable between pH 4.5-9.5. Cruzipain was found to be rapidly and tightly inhibited by various protein inhibitors of the cystatin superfamily (kass = 1.7-79 x 10(6) M-1s-1, Kd = 1.4-72 pM). These results suggest a possible defensive role for the host's cystatins after parasite infection, and may be of use for the design of new therapeutic drugs.

Animals↗
PubMed1992

Cystatins and stefins in ascites fluid from ovarian carcinoma.

Cysteine proteinase inhibitors (CpI) of all three families were found in ascites fluid from patients with ovarian carcinoma. CPIs were isolated by affinity chromatography on carboxymethylated papain Sepharose, followed by gel filtration, anti-stefin-Sepharose and ion exchange chromatography. The highest apparent inhibition against cathepsin B (Cat B) was found in the low molecular mass (LMM) CPI fraction. Immunochemical analysis of this fraction revealed the presence of cystatin C and both stefins A and B while the high molecular mass (HMM) CPI fraction contained kininogens. We demonstrated that CPIs were not completely associated with cysteine proteinases (CPs): about 20% of HMM CPIs and 50% of LMM CPIs were free in native ascites fluid. Affinity chromatography on anti-Cat B-Sepharose revealed that the major LMM CPI, associated with Cat B in native ascites fluid, was the full length form of cystatin C, pI 9.3, and not its truncated form, pI 7.85. The latter was isolated and found to inhibit Cat B in vitro with apparent Ki 0.18 +/- 0.2 nM. Stefin A was isolated from alkaline activated ascites fluid in its two isoforms, pI 4.6 and 4.9. In native ascites, the pI 4.9 isoform was mostly associated with Cat B. Ki for Cat B was 3.55 +/- 1.7 nM, not significantly different from the Ki values measured for stefin A, isolated from other human tissues and biological fluids.

Adult↗
PubMed2006

Cystatin C reference values and aging.

OBJECTIVES: The aim of this study was to determine the reference values for serum cystatin C (CysC) with a particular focus on the effect of aging. DESIGN AND METHODS: The study was performed on a consecutive series of subjects (258 men and 396 women). Laboratory parameters and a detailed personal and family medical history were collected. RESULTS: CysC showed a significant correlation with age in both sexes, which was confirmed with multivariate linear regression after adjustment for SCr (serum creatinine). Age-related reference intervals were established for cystatin C (<45 years, <0.95 mg/L and >45 years, <1.20 mg/L). CONCLUSIONS: The use of CysC reference values adjusted for age should be carefully taken into consideration.

Adult↗
PubMed2005

Genotype (cystatin C) and EEG phenotype in Alzheimer disease and mild cognitive impairment: a multicentric study.

Previous findings demonstrated that haplotype B of CST3, the gene coding for cystatin C, is a recessive risk factor for late-onset Alzheimer's disease (AD; Finckh, U., von der Kammer, H., Velden, J., Michel, T., Andresen, B., Deng, A., Zhang, J., Muller-Thomsen, T., Zuchowski, K., Menzer, G., Mann, U., Papassotiropoulos, A., Heun, R., Zurdel, J., Holst, F., Benussi, L., Stoppe, G., Reiss, J., Miserez, A.R., Staehelin, H.B., Rebeck, G.W., Hyman, B.T., Binetti, G., Hock, C., Growdon, J.H., Nitsch, R.M., 2000. Genetic association of the cystatin C gene with late-onset Alzheimer disease. Arch. Neurol. 57, 1579-1583). In the present multicentric electroencephalographic (EEG) study, we analyzed the effects of CST3 haplotypes on resting cortical rhythmicity in subjects with AD and mild cognitive impairment (MCI) with the hypothesis that sources of resting EEG rhythms are more impaired in carriers of the CST3 B haplotype than non-carriers. We enrolled a population of 84 MCI subjects (42% with the B haplotype) and 65 AD patients (40% with the B haplotype). Resting eyes-closed EEG data were recorded in all subjects. EEG rhythms of interest were delta (2-4 Hz), theta (4-8 Hz), alpha 1 (8-10.5 Hz), alpha 2 (10.5-13 Hz), beta 1 (13-20 Hz), and beta 2 (20-30 Hz). EEG cortical sources were estimated by low-resolution brain electromagnetic tomography (LORETA). Results showed that the amplitude of alpha 1 (parietal, occipital, temporal areas) and alpha 2 (occipital area) was statistically lower in CST3 B carriers than non-carriers (P < 0.01). Whereas there was a trend towards statistical significance that amplitude of occipital delta sources was stronger in CST3 B carriers than in non-carriers. This was true for both MCI and AD subjects. The present findings represent the first demonstration of relationships between the AD genetic risk factor CST3 B and global neurophysiological phenotype (i.e., cortical delta and alpha rhythmicity) in MCI and AD subjects, prompting future genotype-EEG phenotype studies for the early prediction of AD conversion in individual MCI subjects.

Adult↗
PubMed1998

Two-step mechanism of inhibition of cathepsin B by cystatin C due to displacement of the proteinase occluding loop.

Stopped-flow kinetics showed that the inhibition of the lysosomal cysteine proteinase, cathepsin B, by its endogenous inhibitor, cystatin C, occurs by a two-step mechanism, in which an initial, weak interaction is followed by a conformational change. The initial interaction most likely involves binding of the N-terminal region of the inhibitor to the proteinase. Considerable evidence indicates that the subsequent conformational change is due to the inhibitor displacing the occluding loop of the proteinase that partially obscures the active site. The presence of this loop, which allows the enzyme to function as an exopeptidase, thus complicates the inhibition mechanism, rendering cathepsin B much less susceptible than other cysteine proteinases to inhibition by cystatins.

Binding Sites↗
PubMed1995

Cystatin S in secretory granules fractions isolated from submandibular gland of infected rats by Trypanosoma cruzi.

We have measured the relative concentrations of cysteine proteinase in the granular fractions of submandibular glands of control and Trypanosoma cruzi infected rats by using a sensitive solid phase ELISA. Gland samples were homogenized in 0.34 M sucrose and 0.5 mM EDTA in 10 mM HEPES buffer at a pH of 7.4. The extract was centrifuged and filtered through Millipore filters to prepare a purer granular fraction. Immunochemical studies using antibody against cystatin S and electrophoretic analysis showed higher cystatin S levels in infected rats than in control. The role of this inhibitor during acute phase of Chagas disease is discussed.

Acute Disease↗