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PubMed2001

Sympathetic and parasympathetic regulation of cystatin S gene expression.

The autonomic nervous system plays a regulatory role in the differentiation and growth of salivary glands, and in the expression of salivary specific genes. Cystatin S, a member of the evolutionarily conserved family 2 of the cysteine proteinase inhibitor superfamily, expressed in submandibular and parotid glands of rats during development, can be induced in adults by the beta-adrenergic agonist isopreterenol (IPR). It was shown previously that unilateral sympathectomy or bilateral parasympathectomy reduces IPR-induced cystatin S expression. The present experiments demonstrate that IPR-induced cystatin S gene expression in submandibular glands is reduced as early as 3 days post bilateral denervation of both branches of the autonomic nervous system. The reduction is nearly equal to that of either sympathectomy or parasympathectomy alone, suggesting that factor(s) in both sympathetic and parasympathetic fibers are simultaneously required for IPR-induced cystatin S gene expression.

Animals↗
PubMedDate not supplied

Expression of cysteine proteinases and their inhibitor, cystatin beta, in cultured rat mesangial cells.

Matrix expansion in the glomerular mesangial area is observed in diabetic nephropathy. Intracellular breakdown of long-lived proteins was lower in mesangial cells in the high glucose medium than that in the control medium. Enzymatic activity of cathepsin L increased 1.4-fold after 6 h of treatment with the high glucose, and then declined gradually to 72% of control cells after treatment for 36 h. Change in the enzyme activity of cathepsin B showed a similar time course but less magnitude than that of cathepsin L. Immunoblot analysis with anti-cathepsin L antibody showed that change in the enzyme activity of cathepsin L was due to the change in the amount of cathepsin L, and that with anti-cathepsin B antibody showed no change in the amount of cathepsin B in the mesangial cells treated with high glucose. Intracellular cathepsin activities were controlled not only by the amounts but also by the inhibitor cystatin beta. Immunoblot analysis with anti-cystatin beta antibody showed that intracellular levels of cystatin beta increased slightly after 24 h of treatment with high glucose. These changes were derived from changes in mRNA level. These results, therefore, demonstrated that the decrease of intracellular protein breakdown in mesangial cells treated with high glucose medium was due to both suppression of cathepsins and increase of cystatin beta.

Animals↗
PubMed2003

Upregulation of cystatin M during the progression of oropharyngeal squamous cell carcinoma from primary tumor to metastasis.

To identify metastasis-associated molecules in oropharyngeal squamous cell carcinomas (OSCC), we recently compared mRNA expression profiles of cell lines derived from primary and metastatic lesions of OSCC using microarray technology. Cystatin M, an endogenous cathepsin B inhibitor, was expressed 40-fold higher in the metastatic versus the primary tumor cell line. To show that different cystatin expression levels affect the cell lines' sensitivities to TNF-induced apoptosis by differentially regulating cathepsin B activity. The 686Tu and 686Ln cell lines were established from a 49-year-old male patient with an OSCC involving the posterior tongue and oro-pharynx (tumor stage T(3)N(3B)). RT-PCR, Western blots, immunohistochemistry, and in situ hybridization all confirmed increased cystatin M expression in 686Ln compared to 686Tu cells, and in the parent archival tumors. TNF-alpha induced apoptosis was easily detected in 686Tu, but only marginally in 686Ln cells. Thus, we propose that elevated cystatin M expression aids metastasis by blocking intrinsic cathepsin B activity and rescuing tumor cells from TNF-induced apoptosis.

Apoptosis↗
PubMed2002

Enhancement of proteinase inhibitory activity of recombinant human cystatin C using random-centroid optimization.

We had previously written a random-centroid optimization computer program for genetics (RCG) to optimize protein engineering, which was successfully applied to modify single site of the 16 amino acid residues at the active site of B. stearothermophilys neutral protease for improving thermostability [J. Agric. Food Chem., 46 (1998) 1655]. The same program was applied in this study to double-site mutation of the entire sequence of human cystatin C (HCC) with 120 residues for improving its protease inhibitory activity. The RCG program selected two sites simultaneously and amino acid residues to replace the sites selected in the sequence in order to find the best papain-inhibitory activity and stability of the protease inhibitor. Twenty-three double mutants and twenty-two single mutants were expressed by Pichia pastoris. Of the total 45 mutants, G12W/H86V mutant showed a 5-fold increase in the bioactivity over the recombinant wild-type (WT) cystatin. Also, P13F mutant exhibited a half-life temperature (T1/2) 5.2 degrees C higher than 68.2 degrees C of WT in addition to a 56% greater papain inhibitory activity. Mutation for diminishing beta-sheet content reduced polymerization of cystatin C, thus improving papain-inhibitory activity. The approach using RCG was able to improve the functional properties of cystatin by least relying on the prior knowledge of its molecular structure.

Animals↗
PubMed2001

Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping.

The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the 'open interface' of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe 'conformational disease' is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains.

Amino Acid Sequence↗
PubMed2004

Transcriptomic analysis in the leech Theromyzon tessulatum: involvement of cystatin B in innate immunity.

At the present time, there is little information on mechanisms of innate immunity in invertebrate groups other than insects, especially annelids. In the present study, we have performed a transcriptomic study of the immune response in the leech Theromyzon tessulatum after bacterial challenge, by a combination of differential display RT (reverse transcriptase)-PCR and cDNA microarrays. The results show relevant modulations concerning several known and unknown genes. Indeed, threonine deaminase, malate dehydrogenase, cystatin B, polyadenylate-binding protein and alpha-tubulin-like genes are up-regulated after immunostimulation. We focused on cystatin B (stefin B), which is an inhibitor of cysteine proteinases involved in the vertebrate immune response. We have cloned the full-length cDNA and named the T. tessulatum gene as Tt-cysb. Main structural features of cystatins were identified in the derived amino acid sequence of Tt-cysb cDNA; namely, a glycine residue in the N-terminus and a consensus sequence of Gln-Xaa-Val-Xaa-Gly (QXVXG) corresponding to the catalytic site. Moreover, Tt-cysb is the first cystatin B gene characterized in invertebrates. We have determined by in situ hybridization and immunocytochemistry that Tt-cysb is only expressed in large coelomic cells. In addition, this analysis confirmed that Tt-cysb is up-regulated after bacterial challenge, and that increased expression occurs only in coelomic cells. These data demonstrate that the innate immune response in the leech involves a cysteine proteinase inhibitor that is not found in ecdysozoan models, such as Drosophila melanogaster or Caenorhabditis elegans, and so underlines the great need for information about innate immunity mechanisms in different invertebrate groups.

Amino Acid Sequence↗
PubMed1986

Human low-Mr kininogen contains three copies of a cystatin sequence that are divergent in structure and in inhibitory activity for cysteine proteinases.

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.

Amino Acid Sequence↗
PubMed2004

Serum cystatin C for assessment of glomerular filtration rate in hypertensive disorders of pregnancy.

BACKGROUND: Assessment of renal function is important in the evaluation of the pregnant hypertensive patient. The aim of this study was to evaluate the diagnostic utility of cystatin C as a marker of glomerular filtration rate in hypertensive disorders of pregnancy and to correlate this with serum creatinine and creatinine clearance. METHODS: For our study, 198 women who presented with hypertension during pregnancy were recruited at the antenatal clinic of King Edward VIII Hospital in Durban, South Africa, a tertiary referral centre. Exclusion criteria included women with eclampsia, urinary tract infection and chronic renal disease. Routine dipstick urinalysis (Bayer) was performed by midwives to classify patients suffering from the different forms of hypertensive disorders of pregnancy and venous blood samples for determination of serum cystatin C and serum creatinine were collected. The 24-hour urine creatinine clearance was used as the "gold standard" for evaluation of glomerular filtration rate due to concerns of radiation exposure to pregnant women with the use of radio nucleotide markers. RESULTS: The results of testing the 198 women, 72 of whom had preeclampsia, were analyzed. Serum cystatin C showed a significant correlation with creatinine clearance at the 0.01 level (2-tailed) with an r-value of -0.311. Serum creatinine showed a significant correlation with creatinine clearance at the 0.01 level (2-tailed) with an r-value of -0.486. CONCLUSION: Serum cystatin C seems to reflect glomerular filtration rate reliably in hypertensive pregnant women and avoids the inaccuracy associated with the 24-hour urine collection, which is time consuming and subject to improper collection.

Adult↗
PubMed1989

The use of monoclonal antibodies to define levels of cystatin C in normal human serum.

We established four hybridoma cell lines which secreted monoclonal antibodies against human cystatin C. These monoclonal antibodies were of IgA(kappa), IgG2a(lambda), IgG1(kappa), and IgG1(lambda) isotypes, respectively. The association constants (Ka) of the three IgG monoclonal antibodies ranged from 3.6 x 10(9) M-1 to 7.3 x 10(10) M-1. An ELISA technique for the measurement of cystatin C was established by using one of these monoclonal antibodies. The assay procedure was highly specific, sensitive, and reproducible. The lower detectable limit of cystatin C by this procedure was 1.9 ng/ml. The level of cystatin C in normal human serum was 1.16 +/- 0.91 micrograms/ml (mean +/- S.D., n = 274).

Animals↗
PubMed2006

Cystatin C and measures of physical function in elderly adults: the Health, Aging, and Body Composition (HABC) Study.

Most studies of the relation between kidney function and physical function have been conducted in persons with advanced kidney disease and have used creatinine-based measures of kidney function. Cystatin C concentration is a measure of kidney function that is independent of muscle mass, unlike creatinine. Using baseline data on 3,043 elderly adults from the Health, Aging, and Body Composition Study (Blacks and Whites recruited from Pittsburgh, Pennsylvania, and Memphis, Tennessee, in 1997-1998), the authors examined the cross-sectional association between cystatin C level and performance on several tests of physical function. After adjustment for demographic and lifestyle variables, chronic health conditions, and inflammation, each standard-deviation (0.34 mg/liter) increase in cystatin C concentration was associated with 1.32 odds (95% confidence interval (CI): 1.20, 1.46) of not completing a 400-m walk, a 10.9-second (95% CI: 8.1, 13.8) slower 400-m walk time, a 0.11-point (95% CI: 0.09, 0.13) reduction in lower extremity performance score, a 1.12-kg (95% CI: 0.83, 1.40) lower grip strength, and a 4.7-nm (95% CI: 3.5, 5.9) lower knee extension strength. In contrast, when kidney function was measured by estimated glomerular filtration rate, the association of kidney function with physical function was only evident below 60 ml/minute/1.73 m2. In these older adults, mild decrements in kidney function, as measured by cystatin C concentration, were associated with poorer physical function.

Aged↗
PubMed1999

High-dose aprotinin with gentamicin-vancomycin antibiotic prophylaxis increases blood concentrations of creatinine and cystatin C in patients undergoing coronary artery bypass grafting.

Both aprotinin and gentamicin-vancomycin antibiotic prophylaxis have been used widely in cardiac surgery to prevent bleeding and infections, respectively. As the drugs are excreted almost entirely by glomerular filtration, we investigated their action on renal function when administered either separately or together. To increase consistency, we measured serum concentrations of creatinine and cystatin C, a new marker of glomerular filtration rate, that many recent studies have shown to be more sensitive than serum creatinine. One hundred patients undergoing coronary artery bypass surgery were allocated randomly to one of four groups: group A received antibiotic prophylaxis with cefamandole and no aprotinin; group B received cefamandole and high-dose aprotinin; group C received antibiotic prophylaxis with gentamicin and vancomycin, but no aprotinin; and group D received both high-dose aprotinin and gentamicin-vancomycin antibiotic prophylaxis. Data from 84 patients, for whom data collection was complete, were analysed. In the first week after operation, mean serum concentrations of cystatin C and creatinine either remained constant or decreased slowly in all groups, except for group D. In group D, both markers increased gradually from postoperative day 2 onwards. The increase in cystatin C was significant on postoperative day 5 (from mean 1.02 (SD 0.11) mg litre-1 before operation to 1.35 (0.32) mg litre-1; P < 0.05), reaching a peak on postoperative day 7 (1.45 (0.35) mg litre-1; P < 0.05), while the increase in creatinine concentration was significant on postoperative day 6 (from 1.05 (0.16) mg dl-1 before operation to 1.29 (0.34) mg dl-1; P < 0.05). We conclude that simultaneous administration of high-dose aprotinin and prophylactic use of gentamicin with vancomycin increased serum concentrations of cystatin C and creatinine in the first postoperative week in patients undergoing cardiac surgery.

Aged↗
PubMed2002

New insights into the molecular basis of progressive myoclonus epilepsy: a multiprotein complex with cystatin B.

Cystatin B is an anti-proteolytic polypeptide implicated in progressive myoclonus epilepsy (EPM1), a degenerative disease of the central nervous system. The knock-out mouse model of the disease shows apoptosis of the cerebellar granule cells. We have identified five recombinant proteins interacting with cystatin B and none of them is a protease. We show that three of these proteins (RACK-1, beta-spectrin and NF-L) co-immunoprecipitate with cystatin B in rat cerebellum. Confocal immunofluorescence analysis shows that the same proteins are present in the granule cells of developing cerebellum, as well as in Purkinje cells of adult rat cerebellum. We propose that a cystatin B multiprotein complex has a specific cerebellar function and that the loss of this function might contribute to the disease in EPM1 patients.

Animals↗
PubMed2004

Evidence that unrestricted legumain activity is involved in disturbed epidermal cornification in cystatin M/E deficient mice.

Homozygosity for Cst6 null alleles causes the phenotype of the ichq mouse, which is a model for human harlequin ichthyosis (OMIM 242500), a genetically heterogeneous group of keratinization disorders. Here we report evidence for the mechanism by which deficiency of the cysteine protease inhibitor cystatin M/E (the Cst6 gene product) leads to disturbed cornification, impaired barrier function and dehydration. Absence of cystatin M/E causes unrestricted activity of its target protease legumain in hair follicles and epidermis, which is the exact location where cystatin M/E is normally expressed. Analysis of stratum corneum proteins revealed a strong decrease of soluble loricrin monomers in skin extracts of ichq mice, although normal levels of loricrin were present in the stratum granulosum and stratum corneum of ichq mice, as shown by immunohistochemistry. This suggested a premature or enhanced crosslinking of loricrin monomers in ichq mice by transglutaminase 3 (TGase 3). In these mice, we indeed found strongly increased levels of TGase 3 that was processed into its activated 30 and 47 kDa subunits, compared to wild-type mice. This study shows that cystatin M/E and legumain form a functional dyad in epidermis in vivo. Disturbance of this protease-antiprotease balance causes increased enzyme activity of TGase 3 that could explain the observed abnormal cornification.

Animals↗
PubMed1985

Serum concentration of cystatin C, factor D and beta 2-microglobulin as a measure of glomerular filtration rate.

Serum concentrations of creatinine and of the three low molecular weight (LMW) proteins cystatin C, factor D of the complement system and beta 2-microglobulin were measured in 135 consecutive patients, whose glomerular filtration rates (GFR) were determined by Cr-EDTA. In the total patient series, the reciprocals of S-creatinine and S-cystatin C were numerically and, in males, significantly more closely correlated to GFR than the reciprocals of S-factor D. The reciprocals of beta 2-microglobulin showed a weaker correlation to GFR than those of the other three substances. The calculated glomerular elimination rates of creatinine, cystatin C and factor D were normally distributed, in contrast to those of beta 2-microglobulin. According to data presented so far, cystatin C seems to be the LMW protein of first choice when GFR is to be estimated by measuring the plasma concentration of a LMW protein.

Adolescent↗
PubMed2006

Hereditary cystatin C amyloid angiopathy: genetic, clinical, and pathological aspects.

Hereditary cystatin C amyloid angiopathy (HCCAA) is a rare, fatal amyloid disease in young people in Iceland caused by a mutation in cystatin C, which is an inhibitor of several cysteine proteinases, such as cathepsins S, B, and K. The same mutation in cystatin C, L68Q, has been found in all patients examined so far pointing to a common founder. Most of the families can be traced to a region in the northwest of Iceland, around Breidafjordur bay. Mutated cystatin C forms amyloid, predominantly in brain arteries and arterioles, but also to a lesser degree in tissues outside the central nervous system such as skin, lymph nodes, testis, spleen, submandibular salivary glands, and adrenal cortex. The amyloid deposition in the vessel walls causes thickening of the walls leading to occlusion or rupture and resulting in brain hemorrhage. Although the amyloid can be detected outside the brain, the clinical manifestation is restricted to the brain, and usually consists of repeated hemorrhages leading to paralysis. Sometimes the initial signs of hemorrhage are dementia and personality changes.

Adolescent↗
PubMed2006

The role of cystatin C in cerebral amyloid angiopathy and stroke: cell biology and animal models.

A variant of the cysteine protease inhibitor, cystatin C, forms amyloid deposited in the cerebral vasculature of patients with hereditary cerebral hemorrhage with amyloidosis, Icelandic type (HCHWA-I), leading to cerebral hemorrhages early in life. However, cystatin C is also implicated in neuronal degenerative diseases in which it does not form the amyloid protein, such as Alzheimer disease (AD). Accumulating data suggest involvement of cystatin C in the pathogenic processes leading to amyloid deposition in cerebral vasculature and most significantly to cerebral hemorrhage in patients with cerebral amyloid angiopathy (CAA). This review focuses on cell culture and animal models used to study the role of cystatin C in these processes.

Alzheimer Disease↗
PubMed1990

Cystatin C, a human proteinase inhibitor, blocks replication of herpes simplex virus.

Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids. Cystatin C and a tripeptide derivative (Z-LVG-CHN2) that mimics its proteinase-binding center, were tested for possible antiviral activity against herpes simplex virus type 1 (HSV) and poliovirus type 1. Both recombinant cystatin C and Z-LVG-CHN2 displayed strong inhibitory effects on HSV replication, whereas no significant effect on poliovirus replication was seen. The molar concentration of cystatin C that gave total inhibition of HSV replication was lower than that of either Z-LVG-CHN2 or of acyclovir, the drug currently most used against HSV infections. These results suggest that cysteine proteinase inhibitors might play a physiological role as inhibitors of viral replication and that such proteinase inhibitors, or peptide derivatives that mimic their proteinase-binding centers, might be used as antiviral agents.

Amino Acid Sequence↗
PubMedDate not supplied

Creatinine clearance, Cockcroft-Gault formula and cystatin C: estimators of true glomerular filtration rate in the elderly?

BACKGROUND: The aim of this study was to assess the accuracy and precision of estimators of true glomerular filtration rate (GFR) (Cockcroft formula, measured creatinine clearance (CCR) and a cystatin-C-based estimation) in elderly patients attending a geriatric department. Additionally, parameters influencing GFR in the elderly were evaluated. METHODS: 30 patients aged 57-90 years treated in the Geriatric Department for pulmonary or cerebral diseases were included in the study. Nine patients were diabetic and 16 hypertensive. Exclusion criteria were advanced dementia, acute heart failure and primary renal disease. Inulin clearance (CINU), CCR and estimation by Cockcroft-Gault equation (CG) were performed on the same day. For comparison of the methods an analysis according to Bland and Altman was used, depicting the mean difference between the methods and the limits of agreement of the differences, representing their 95% interval of confidence. Furthermore, the influence of confounding variables on GFR estimation was analyzed by multiple regression. RESULTS: Baseline characteristics showed a median age of 74.5 years and a median body weight of 66.7 kg. Median values for serum creatinine 88.4 micromol/l, 5.74 mmol/l for urea and 1.57 mg/l for cystatin C. CCR (median: 51.6 ml/min) and CG (median: 63.0 ml/min) underestimated CINU (median: 83.3 ml/min). Both methods showed poor precision compared with CINU. The upper limit of agreement of the difference was 101.3 ml/min for CCR and 81.4 ml/min for CG, the lower limit was -33.8 ml/min for CCR and -24.6 ml/min for CG. Among frequently used variables to predict GFR, the reciprocal of serum creatinine and body weight revealed a significant influence but not age or gender. A cystatin-C-based estimation of GFR, derived from regression analysis, did not improve the precision of the estimation of GFR compared to CG. Additionally, the occurrence of diabetes mellitus disclosed a borderline influence on the estimation of GFR. CONCLUSION: CCR is not only inconvenient and time consuming, but also imprecise and inaccurate in the elderly, mainly due to reduced muscle mass and erroneous urine sampling. CG and a cystatin-C-based estimation are slightly more adequate, but overall there is no sufficiently precise formula for GFR estimation in the elderly.

Aged↗