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The association of a cystatin C gene polymorphism with late-onset Alzheimer's disease and vascular dementia.

A polymorphism in the cystatin C (CST3) gene was suggested to associate with Alzheimer's disease (AD). In the present study we attempted to determine the association between CST3 polymorphism and AD or vascular dementia (VD), and whether such effects are dependent of the APOE4 allele. The polymorphisms of CST3 genotype were determined using polymerase chain reactions (PCR) followed by gel electrophoresis in 124 AD, 70 VD, and 115 control individuals. No statistical difference in CST3B allele frequencies was observed among all three groups. Associations between CST3B/B genotype and AD patients older than 75-year-old, or VD patients younger than 75-year-old were evident. The APOE4 allele alone significantly increased the odds for the developing AD, but not VD. A logistic regression analysis revealed that either CST3 or its interaction with APOE4 were not significant predictors of AD. However, a synergistic association of CST3 and APOE4 alleles was observed in predicting VD patients. These results suggest that CST3 might interact with APOE4 on conferring vascular pathologies.

Aged↗

Cystatin C antagonizes transforming growth factor beta signaling in normal and cancer cells.

Cystatin C (CystC) is a secreted cysteine protease inhibitor that regulates bone resorption, neutrophil chemotaxis, and tissue inflammation, as well as resistance to bacterial and viral infections. CystC is ubiquitously expressed and present in most bodily fluids where it inhibits the activities of cathepsins, a family of cysteine proteases that can promote cancer cell invasion and metastasis. Transforming growth factor beta (TGF-beta) is a multifunctional cytokine endowed with both tumor-suppressing and tumor-promoting activities. We show herein that TGF-beta treatment up-regulated CystC transcript and protein in murine 3T3-L1 fibroblasts. Moreover, CystC mRNA expression was down-regulated in approximately 50% of human malignancies, particularly cancers of the stomach, uterus, colon, and kidney. Overexpression of CystC in human HT1080 fibrosarcoma cells antagonized their invasion through synthetic basement membranes in part via a cathepsin-dependent pathway. Independent of effects on cathepsin activity, CystC also reduced HT1080 cell gene expression stimulated by TGF-beta. Invasion of 3T3-L1 cells occurred through both cathepsin- and TGF-beta-dependent pathways. Both pathways were blocked by CystC, but only the TGF-beta-dependent pathway was blocked by a CystC mutant (i.e., delta14CystC) that is impaired in its ability to inhibit cathepsin activity. Moreover, CystC and delta14CystC both inhibited 3T3-L1 cell gene expression stimulated by TGF-beta. We further show that CystC antagonized TGF-beta binding to its cell surface receptors, doing so by interacting physically with the TGF-beta type II receptor and antagonizing its binding of TGF-beta. Collectively, our findings have identified CystC as a novel TGF-beta receptor antagonist, as well as a novel CystC-mediated feedback loop that inhibits TGF-beta signaling.

3T3-L1 Cells↗

Effect of antisense peptide binding on the dimerization of human cystatin C--gel electrophoresis and molecular modeling studies.

Human cystatin C (HCC) shows a tendency to dimerize. This process is particularly easy in the case of the L68Q HCC mutant and might lead to formation of amyloid deposits in brain arteries of young adults. Our purpose was to find ligands of monomeric HCC that can prevent its dimerization. Eleven antisense peptide ligands of monomeric HCC were designed and synthesized. The influence of these ligands on HCC dimerization was studied using gel electrophoresis and molecular modeling methods. The results suggest that all the designed peptides interact with monomeric HCC facilitating its dimerization rather than preventing it.

Amino Acid Sequence↗

[Serum cystatin C in haemodialysed patients].

BACKGROUND: Cystatin C (CyC) is protein (m. w. 13 300), which is produced by nucleated cells, filtered through glomeruli and subsequently reabsorbed and degraded in tubuli. OBJECTIVE: To investigate changes in serum CyC concentration during two low-flux membrane haemodialyses (HD-I, HD-II) and relationship between serum CyC a creatinine concentrations. PATIENTS AND METHODS: Serum CyC was determined in 17 patients on chronic haemodialysis during HD-I and HD-II by immunonephelometric method (Dade Behring, Austria). RESULTS: Significant correlation between serum CyC and creatinine before HD-I and before/after HD-II was found (p < 0.001). Serum CyC before HD-I (median 6.3 mg/I, 95 % CI 5.5 - 6.7) increased after HD-I (7.1 mg/l, 6.1 - 8.9) (p < 0.001). Serum CyC before HD-II (6.4 mg/l, 5.8 - 7.2) increased after HD-II (8.0 mg/l, 7.3 - 9) (p < 0.001), while serum creatinine decreased after HD-I and HD-II (p < 0.001). There was correlation between the increase in serum CyC and albumin during HD-I (p < 0.001) and HD-II (p < 0.01). CONCLUSION: There was close correlation between serum CyC and creatinine before haemodialyses. Serum CyC increased after haemodialyses, due to CyC non-dialysability through low-flux membrane and haemoconcentration. Unlike creatininaemia, serum CyC reflects the residual renal function even after haemodialyses.

Adult↗

[Cystatin C, beta2-microglobulin and C-reactive protein in hemodiafiltration and on-line endogenous liquid reinfusion and in low flux polysulphone bicarbonate conventional hemodialysis].

PURPOSE: Dialysis morbidity results partly from middle and large molecule retention, which is poorly removed by conventional hemodialysis (HD). The potential benefit of convective treatments could be the enhanced toxin removal over a wide molecular weight spectrum. This study aimed to evaluate cystatin C (cis), beta2-microglobulin (beta2-m) and C-reactive protein (CRP) removal behavior during hemodiafiltration reinfusion vs conventional low-flux HD (1.8 m2 low-flux polysulphone) (bicarbonate dialysis (BD)). The molecular weights of the substances evaluated in this study were as follows: cis = 13,300 daltons, beta2-m = 11,818 daltons, CRP = 160,000 daltons. METHODS: Twelve patients on stable HD (six males, six females), were enrolled; six patients underwent BD and six patients underwent HFR. We measured arteriovenous serum cis, beta2-m and CRP levels, in three consecutive mid-week sessions at the following periods: pre/post-dialysis and after 60 min from the beginning of the session. At 60, 120 and 180 min of HFR, we collected the ultrafiltrate for cis, beta2-m, and CRP evaluation. RESULTS: Cis, beta2-m and CRP mean values did not differ at pre-dialysis in the two groups. Pre/post- dialysis difference for cis in HFR vs BD was statistically significant (p=0.002) because cis reduced in HFR and increased in BD during the session. Beta2-m and CRP pre/post- dialysis differences in HFR vs BD were not significant. Cis clearance, measured 60 min after the beginning of the session was 34.2 +/- 20.1 mL/min in HFR and 24.8 +/- 18.4 mL/min in BD (p<0.05). beta2-m and CRP clearances did not differ among the treatments. Regarding the ultrafitrate concentrations during the HFR session, cis significantly decreased (2.5 +/- 0.6 mg/dL at 60 min and 2.0 +/- 0.4 mg/dL at 180 min; p=0.004), as well as beta2-m (21.5 +/- 12.9 mg/dL and 19.0 +/- 14.1 mg/dL, respectively; p=0.02). Ultrafiltrate CRP values, as expected, did not differ during HFR. CONCLUSIONS: This study demonstrated that cis, a middle molecule, is well depurated in HFR, while in BD it increases. Beta2-m, although better removed in the convective phase during HFR, does not demonstrate a removal difference in HFR and in BD. CRP, a large molecule, does not have significant removal. Since cis and beta2-m have almost the same molecular weight, why do they have a different depuration? We need further studies to evaluate if membranes can remove these molecules or if protein electrical charges or their stereoscopy enables their removal.

Bicarbonates↗

Can cystatin C be a better marker for the early detection of renal damage in primary hypertensive patients?

In this study, we aimed to compare Cystatin C (Cys C) with other traditional glomerular filtration rate (GFR) markers and to evaluate its superiority over them in detecting early renal involvement in patients with primary hypertension. Fifty-one primary hypertensive patients and 29 healthy control subjects, who were similar in terms of age and gender, were included in the study. In all subjects serum levels of Cys C, beta-2 microglobulin, serum creatinine (SCr), uric acid, BUN, albumin; 24 h urinary levels of protein (Upro), albumin (Ualb) and creatinine were measured. The GFR was calculated according to Creatinine Clearance (CrCl), Cockcroft-Gault (CG) and Modification of Diet in Renal Disease (MDRD) formulas. The MDRD was used as the reference method. A GFR<80 mL/min/1.73 m2 was considered as the lower cut-off limit. Mean levels of the serum parameters were found to be significantly higher in the patient group than they were in the control group (p<0.05). Mean CrCl, CG, and MDRD levels were lower in patients than they were in controls but the difference was statistically significant for CG and MDRD. The serum parameter having the best correlation with MDRD was SCr (r = -0.760) in patients and Cys C (r = -0.622) in controls. However, in ROC analysis; the area under curve (AUC) for Cys C was found to be superior (AUC = 0.900) to the other markers. The CrCl was the parameter having the worst diagnostic efficiency (AUC = 0.598). As a conclusion, compared to other traditional markers, measurement of Cys C may be a better parameter to estimate GFR, especially to detect mild reductions of GFR in primary hypertensive patients.

Adult↗

Serum cystatin C and urinary enzymes as screening markers of renal dysfunction in diabetic patients.

BACKGROUND: In clinical practice, the assessment and follow-up of early renal dysfunction is important in diabetic nephropathy. Therefore, this study was designed to determine whether the serum cystatin C (Cys C) and activities of some tubular enzymes could be used as screening markers for renal dysfunction in diabetic patients. METHODS: Serum Cys C levels and urinary activities of N-acetyl-b-D-glucosaminidase (NAG), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) and whole blood glycolyse hemoglobin (HbA1C) were measured in 56 diabetic patients and 20 healthy subjects (controls). The results were compared with serum creatinine (Cr) and creatinine clearance (CCr), which were measured and estimated with the Cockcroft-Gault formula (CCG) and 24-hr urine microalbuminuria (MAU). We examined the influence of albuminuria, HbA1C and CCr levels of patients on the levels of the analyzed parameters. Sensitivity and specificity for the diagnosis of renal impairment were calculated by a receiver operating characteristics (ROC) curve for serum Cys C, Cr and urinary enzymes. RESULTS: In normoalbuminurics, only serum Cys C levels and urinary NAG activities were found elevated as compared to controls. In addition to the elevation of serum Cys C levels and urinary activities of NAG, urinary ALP and LDH activities were also found elevated in microalbuminurics. Serum Cys C levels and urinary NAG, ALP, LDH activities started to increase above the normal range when CCr declined and while serum Cr was in the normal range in patients with 50<CCr<80 (grade 2 group). Moreover, these parameters in patients with good metabolic control were significantly lower than in patients with poor metabolic control. There was a significant positive correlation between serum Cys C, urinary NAG, LDH, ALP activities and serum Cr levels. However, there was an inverse correlation between serum Cys C, urinary NAG, LDH, ALP and CCr and CCG in diabetic patients. The ROC plot indicated that serum Cys C and urinary NAG and ALP had higher sensitivity than serum Cr and met the criteria for detecting glomerular and tubular dysfunction as screening tests for early diagnosis of diabetic nephropathy. CONCLUSION: This study demonstrated that measuring serum Cys C levels and urinary NAG, ALP and LDH activities could be useful as screening markers to follow-up glomerular and tubular dysfunction in diabetic patients.

Acetylglucosaminidase↗

[Inflammation, atherosclerosis and cardiovascular disease risk: PAPP-A, Lp-PLA2 and cystatin C. New insights or redundant information?].

It is well-known that inflammation plays a role in atherogenesis, atherosclerotic plaque progression, and acute coronary syndrome. Inflammatory cells, and cytokines and other biomolecules are implicated in these processes, and have, therefore, been investigated as potential markers of atherosclerotic plaque progression and cardiovascular disease risk. The best characterized and most widely studied is C-reactive protein. However, its role in the clinical setting is still debated. Emerging novel biomarkers that may provide information complementary to that derived from C-reactive protein include pregnancy-associated plasma protein A, lipoprotein-associated phospholipase A2, and cystatin C. This article focuses on the potential value of these three new markers in patients with coronary heart disease, and their use as markers of disease risk in apparently healthy individuals.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

[Cystatin C as estimator of glomerular filtration rate in patients with advanced chronic renal disease].

Serum cystatin C (CysC) has been shown to be more accurate than serum creatinine (Cr) in estimating renal function, especially in patients with mildmoderate chronic renal failure. However, it is unknown whether CysC provides or not any advantage over Cr in severe chronic renal failure. The aim of this study was to establish the accuracy of CysC in estimating the glomerular filtration rate (GFR) in advanced chronic renal failure patients. The study group consisted of 94 patients (57 females, mean age 61 +/- 16 years) with advanced chronic renal failure. None of them had thyroid dysfunction or was on corticoid treatment. CFR was measured by TC99mDTPA, and simultaneously, serum CysC (particle enhanced immunonephelometry) and Cr (modified Jaffe's kinetic reaction) were also determined. Serum Cr and CysC levels were correlated with GFR, and the influences of age, sex and diabetes on these correlations were analyzed. The predictive value of CysC and Cr to estimate a GFR less than 15 ml/min/1.73 m2 was analyzed by measuring the crea-under-the-curve (AUC) with Receiver-Operating Characteristics (ROC) plots. The mean CFR was 16.49 +/- 4.65 ml/min/1.73 m2. The mean concentrations of Cr and CysC were 4.19 +/- 1.19 mg/L and 3.44 +/- 0.73 mg/L, respectively. Both Cr and CysC correlated significantly with GFR (R = 0.49, p < 0.0001 and R = 0.52, p < 0.0001, respectively). Age and sex influenced on the correlation between Cr and GFR, but these demographic characteristics did not influence on the correlation between CysC and GFR. The AUC for the prediction of a GFR less than 15 ml/min/1.73 m2 with serum Cr was 0.675 (p = 0.004), while with CysC was 0.633 (p = 0.030). In conclusion, both serum Cr and CysC are highly inaccurate markers of renal function in advanced chronic renal failure patients.

Biomarkers↗

Gene mapping of human cathepsins and cystatins.

Chromosomal locations of human cathepsin B, stefin A and kininogen have been determined. Using techniques of somatic cell hybrids and polymerase chain reaction, cathepsin B has been mapped to 8p22, stefin A to 3cen-q21, and kininogen to 3q26-qter. Comparing this data with currently available mapping loci, it can be concluded that many members of cathepsins are dispersed in different chromosomes, whereas members of the cystatin family are restricted to only two human chromosomes, 3 and 20.

Cathepsin B↗

Characterization of monoclonal antibodies to human salivary (glyco) proteins. Cellular localization of mucin, cystatin-like 14 kD protein and 20 kD glycoprotein in the human submandibular gland.

Using the hybridoma technique, monoclonal antibodies (Mabs) have been produced against three different types of human salivary proteins: high molecular weight mucin, a 20 kD glycoprotein and a 14 kD protein, identified as a member of the cystatin family. The Mabs appeared to be highly specific to their antigen in Elisa and immunoblotting tests. The Mabs were of the IgG-1 (against 20 kD glycoprotein) and IgM (against 14 kD protein and mucin) type. For the 14 kD protein and the 20 kD glycoprotein it was demonstrated that they are present mainly in submandibular-sublingual saliva. None of the antigens studied could be localized distinctly in the human parotid gland. In the submandibular gland, the three proteins have a different pattern of localization. The mucins have been detected particularly in the apical part of the mucous acinar cells, the 20 kD glycoprotein mainly in the serous acinar cells and the 14 kD protein in both serous acinar cells and striated duct cells.

Antibodies, Monoclonal↗

Chemical synthesis of a gene for human cystatin C and its expression in E. coli.

A DNA containing the coding sequence for the human cysteine proteinase inhibitor protein cystatin C has been obtained by enzymatic ligation of chemically synthesized deoxyoligonucleotides, using the Khorana ligation method. The 375 bp synthetic gene carries signals for the translation initiation and termination and was expressed in E. coli as a beta-galactosidase fusion protein as well as a secreted protein under the control of the E. coli alkaline phosphatase signal sequence. The secreted hybrid protein was shown to have similar biological properties as the authentic protein isolated from human plasma.

Amino Acid Sequence↗

Correlation of plasma concentrations of cystatin C and creatinine to inulin clearance in a pediatric population.

Measurement of blood concentrations of cystatin C (cysC), a cysteine protease inhibitor present in human plasma, has been suggested for use as an indicator of glomerular filtration rate (GFR) in a manner analogous to the use of plasma creatinine (SCR). In this study, cysC and SCR were measured in plasma from pediatric patients (4-19 years) with renal disease for whom a "gold standard" measurement of GFR via inulin clearance (C(IN)) was available. The data analyses were divided into two age groups: group A (4-12 years, n = 26) and group B (12-19 years, n = 34). For both age groups, the linear correlation coefficient of [cysC](-1) vs C(IN) (mL/min/1.73 m2) (r = 0.765 for group A and r = 0.869 for group B) was less than that of the linear correlation coefficient of [SCR](-1) vs C(IN) (r = 0.841 for group A and r = 0.892 for group B). As a single measurement for detection of abnormal GFR, however, the optimum receiver-operator characteristic point for cysC measurement (for group A at cysC >1.2 mg/L, sensitivity = 80%, specificity = 91%; and for group B at cysC >1.4 mg/L, sensitivity = 87%, specificity = 100%) was numerically superior to that for SCR measurement (for group A at SCR >8.0 mg/L, sensitivity = 67%, specificity = 100%; and for group B at SCR >9.0 mg/L, sensitivity = 91%, specificity = 91%), using a reference value for normal GFR of C(IN) > 90 mL/min/1.73 m2. However, these differences were not statistically significant. CysC measurement appears to be broadly equivalent to SCR measurement for estimation of GFR in pediatric patients.

Adolescent↗

Identification of acid cysteine proteinase inhibitor (cystatin A) in the human thymus.

BACKGROUND: Acid cysteine proteinase inhibitor (ACPI, also called cystatin A) is a protein that is present in the epithelial cells of the skin and in the dendritic reticulum cells of lymphoid tissues. In this study the presence and cellular localization of ACPI in the thymus was investigated. METHODS: The cellular and topographical location of ACPI was immunohistochemically demonstrated in the normal thymus of man. RESULTS: ACPI was found in the cells of the Hassall's corpuscles and in many medullary cells. Most of these cells were epithelial cells, as shown by the results of immunohistochemical cytokeratin and epithelial membrane antigen stainings. Also, some individual cytokeratin negative but S-100 positive medullary reticular dendritic cells were stained with ACPI. CONCLUSIONS: The finding that ACPI is constantly present in the thymus at restricted and specific cellular locations leads to the suggestion that protease inhibitors may play a role in specific thymic functions.

Antibodies↗

Fermenter production of an artificial fab fragment, rationally designed for the antigen cystatin, and its optimized crystallization through constant domain shuffling.

The synthetic antibody model "M41" was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine C kappa and CH1 gamma 1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P2(1)2(1)2(1) with unit cell dimensions a = 104.7 A, b = 113.9 A, c = 98.8 A and diffracted X-rays to a nominal resolution of 2.5 A.

Amino Acid Sequence↗

Direct mapping of seven genes encoding human type 2 cystatins to a single site located at 20p11.2.

The human type 2 cystatins are encoded by a multigene family of eight to nine members. Five functional genes (CST1-CST5) and two pseudogenes (CSTP1 and CSTP2) have previously been cloned. We have developed a panel of short genomic fragments that serve as gene-specific probes for these seven genes under hybridization conditions used for Southern blots or fluorescence in situ hybridization (FISH). Analysis of BssHII-digested genomic DNA using these probes was consistent with association of all seven genes with a 905-kb fragment, suggesting physical clustering at a single site. The cluster was assigned to chromosome 20 by screening a somatic cell hybrid panel with an STS for CST2. This assignment was confirmed for each gene by FISH using repeat-free gene-specific genomic probes ranging in size from 2.3 to 0.8 kb. All the loci mapped to 20p11.2.

Animals↗

Walking, cloning, and mapping with YACs in 3q27: localization of five ESTs including three members of the cystatin gene family and identification of CpG islands.

Using yeast artificial chromosomes, we have generated a high-resolution physical map for 2.7 Mb of human chromosomal region 3q27. The YAC clones group into three contigs, one of which has also been linked to the CEPH YAC contig map of human chromosome 3. Fluorescence in sity hybridization has been used to order the contigs on the chromosome and to estimate the distance between them. Expressed sequence tags for five genes, including three members of the cystatin gene family and a gene thought to be involved in B-cell non-Hodgkin lymphoma, have been placed within the YAC contigs, and 12 putative CpG islands have been identified. These YACs provide a useful resource to complete the physical mapping of 3q27 and to begin identification and characterization of further genes that are located there.

Base Composition↗

The rational construction of an antibody against cystatin: lessons from the crystal structure of an artificial Fab fragment.

In a protein design study the artificial antibody M41 was modelled with its binding site complementary to the protease inhibitor cystatin, which was chosen as a structurally well-characterized "antigen". The modelling of M41 took advantage of the crystal structure of the anti-lysozyme antibody HyHEL-10 as a structural template. Its combining site was reshaped by replacing 19 amino acid side-chains in the hypervariable loops. In addition, ten amino acid residues were substituted in the framework regions. The crystal structure of the corresponding antibody model M41, which was produced as an F(ab) fragment in Escherichia coli, was determined at a resolution of 1.95 A. The crystals exhibited symmetry of the space group P2(1)2(1)2(1) (a = 96.5 A; b = 103.5 A; c = 113.6 A) with two F(ab) fragments in the asymmetric unit, which were independently refined (final R-factor 21.7%). The resulting coordinates were used for a detailed comparison with the modelled protein structure. It was found that the mutual arrangement of the six complementarity-determining regions as well as most of their backbone conformation had been correctly predicted. One major difference that was detected for the conformation of a five residue insertion in complementarity-determining region L1 could be explained by an erroneously defined segment in the structure of the antibody 4-4-20, which had been used as a template for this loop. In the light of more recent crystallographic data it appears that this segment adopts a new canonical structure. Apart from this region, most of the side-chains in the antigen-binding site had been properly placed in the M41 model. There was however one important exception concerning Trp H98, whose side-chain conformation had been kept as it appeared in HyHEL-10. The differing orientation of this residue in the model compared with the crystal structure of the artificial F(ab) fragment M41 explains why an antigen affinity could not be detected so far. The detailed analysis of this and other, more subtle deviations suggests how to make this F(ab) fragment function by introducing a few additional amino acid changes into M41.

Amino Acid Sequence↗