Is cystatin C more effective than creatinine in predicting adverse cardiovascular outcomes in elderly people?
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Nomenclature and classification of the proteins homologous with the cysteine-proteinase inhibitor chicken cystatin.
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Cysteine Proteinase Inhibitors↗
A cystatin-like cysteine proteinase inhibitor from venom of the African puff adder (Bitis arietans).
Venoms from eight snakes have been screened for inhibitory activity against papain, strong activity being found in that of the African puff adder, Bitis arietans. The inhibitor from B. arietans venom has been purified by affinity chromatography on carboxymethyl-papain-Sepharose and ion-exchange chromatography. The inhibitor had an apparent Mr of 13,000 in SDS/polyacrylamide gel electrophoresis, and pI value of 6.5 (major component) or 6.3 (minor component). Values of Ki for the inhibition of papain, cathepsin B and dipeptidyl peptidase I were 0.10, 2.7 and 0.23 nM, respectively; chicken calpain was not inhibited.
Animals↗
Role for cathepsin B and cystatins in tumor growth and progression.
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Cathepsin B↗
Cysteine proteinase inhibitor level in tumor and normal tissues in control and cured mice.
Cystatin C is the best known extracellular endogenous cysteine proteinase inhibitor and has been studied as a possible index of tumor growth and as a marker of the effectiveness of antitumor therapy. The aim of this study was to evaluate cystatin C concentrations in murine tumor tissues (compared with other organs not directly involved with tumor development, such as the liver and spleen) during treatment with several antitumor drugs (Ukrain and/or cyclophosphane). Cystatin C concentrations in murine tissues and biological fluids was determined by enzyme-linked immunosorbent (ELISA) assay. The cystatin C ELISA test is a sandwich immunoassay, which uses immobilized rabbit antihuman cystatin C Pab and mouse antihuman cystatin C Mab-HRP (monoclonal antibodies, conjugated with horseradish peroxidase). We observed decreased serum cystatin C concentrations compared with controls in all nontreated tumor models: HA-1 hepatoma (solid and ascitic forms), lung adenocarcinoma (solid and ascitic forms) and LS lymphosarcoma. In the ascitic fluid of mice with HA-1 hepatoma the cystatin C concentration was much lower than in the serum of the same mice (about 20-fold lower). In the HA-1 model of hepatoma cells cystatin C concentration decreased about 2-3-fold compared with the control (intact liver) and Ukrain significantly increased the cystatin C concentration. Cyclophosphane treatment of LS lymphosarcoma significantly increased the cystatin C concentration in serum. Cyclophosphane treatment (50 mg/kg, single injection) increased cystatin C by up to 8-fold more in tumor issue. Ukrain treatment of LS lymphosarcoma was also followed by increased levels of cystatin C in tumor tissue (4-fold); cyclophosphane plus Ukrain had a similar positive effect. In the group with LS lymphosarcoma Ukrain or cyclophosphane plus Ukrain treatment induced a significant increase in cystatin C concentration in liver. Liver cystatin C concentration decreased in the HA-1 hepatoma group and treatment with Ukrain or carboxymethylated beta-1, 3-glucan (CMG) increased this index in both groups. Spleen cystatin C concentrations decreased about 5-fold in LS lymphosarcoma compared with controls and combined treatment with cyclophosphane plus Ukrain restored the index to the normal value. We can conclude that both murine tumors studied were characterized by low cystatin C concentrations in tumor tissues and decreased cystatin C concentrations (to a lesser degree) were also observed in liver and spleen as a result of the "toxic" effect of tumor bearing. Effective treatment in all cases (especially with Ukrain or a combination of cyclophosphane plus Ukrain) induced a significant increase in cystatin C. Obviously, the decrease in cystatin C concentration predominantly in tumor tissue was connected with tumor development and restoration of cystatin C level may be used as a marker of efficacy of antitumor therapy.
Alkaloids↗
Effect of periodontal treatment on the protein composition of whole and parotid saliva.
Cystatins are physiological inhibitors of cysteine proteinases and widely distributed in human tissues and fluids including saliva. Cystatins S, SA, SN, and D are only found in glandular saliva and tear fluid whereas cystatin C has been detected in all tested biological fluids. Previous studies demonstrated that total cystatin activity and cystatin C concentration were highest in whole and parotid saliva of periodontitis patients compared to healthy subjects suggesting a response of the salivary glands to an inflammatory condition of the oral cavity. Based on these results we studied a possible change in the concentration of cystatin S, cystatin C, albumin, IgA, amylase activity, and cystatin activity in a whole and parotid saliva of 20 periodontitis patients as a consequence of periodontal treatment. Saliva samples were quantified for cystatins S and C, albumin, and IgA by enzyme-linked immunosorbent assay. Amylase was determined in an activity assay and total cystatin activity was measured towards pa-pain. The clinical condition of the subjects improved significantly after 6 months of periodontal therapy whereas biochemical analyses of whole and parotid saliva indicated that significant changes in salivary protein composition occurred only in whole saliva. Total cystatin activity (P < 0.05) and cystatin C concentration (P < 0.05) of whole saliva samples collected after periodontal treatment decreased to normal healthy control values. Further, concentrations of cystatin S were unchanged during the periodontal treatment process. These results suggest that other sources of cystatins than the parotid gland i.e.; other salivary glands or crevicular fluid, are involved in the decrease of total cystatin activity in whole saliva after periodontal treatment.
Adult↗
Cysteine proteinase inhibitor in cultured human medullary thyroid carcinoma cells.
The TT cell line of human medullary thyroid carcinoma, that retains some of the differentiated functions of thyroid C cells including the synthesis and secretion of calcitonin, was found to contain and release into the culture medium cysteine proteinase inhibitor(s), cystatin(s). The major inhibitor, which is similar to, if not identical with, cystatin C, is constitutively released, or secreted, by TT cells. The rate of secretion of cystatin, quantified by titration of inhibition of papain, was stimulated by dibutyryladenosine 3':5'-cyclic monophosphate, forskolin, the calcium ionophore A 23187, and by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Neither forskolin nor TPA had, however, an effect on the level of the inhibitor in TT cells. Treatment with n-butyrate strongly inhibited the proliferation of TT cells, and led, in 4 to 7 days, to a doubling of the intracellular concentration of cystatins. Northern blot hybridizations to a 32P-labeled riboprobe complementary to human cystatin C cDNA indicated that cAMP, forskolin, and TPA had no effect on the steady-state levels of cystatin C mRNA. These data indicate that release of cystatin(s) from TT cells is regulated by cAMP-calcium-protein kinase C mechanisms that appear to be similar to those that regulate the secretion of calcitonin from these cells. However, in contrast to the calcitonin gene, the expression of the cystatin C gene in these cells is not regulated by cAMP or TPA. By a combination of acetone fractionation, affinity chromatography on Cm-papain-Sepharose, and gel exclusion chromatography a protein of approximately 14 kilodaltons was isolated from TT cells that reacted with antibodies against human cystatin C, and strongly inhibited papain. Cystain secreted by TT cells also had a molecular weight of 14 kilodaltons, and reacted with anti-human cystatin C antibodies. The physiologic and pathologic roles of cystatins in different cell types remain to be established. The TT cells provide a suitable cell type to study the regulation of the expression of the cystatin gene and the mechanism of cystatin release.
Blotting, Northern↗
Molecular basis for amyloidosis related to hereditary brain hemorrhage.
The aim of the project has been to elucidate molecular events leading to amyloidosis in Hereditary Cystatin C Amyloid Angiopathy (HCCAA) patients, to enable simple diagnosis of the disease and with the ultimate goal to understand the amyloid formation process in detail, in order to develop inhibitors to the process. At the DNA level, a point mutation segregating with HCCAA was identified in the cystatin C gene on chromosome 20, after basic characterization of cDNA and gene for the wildtype protein. The mutation results in the amino acid substitution Leu-68-Gin (L68Q) and abolishes a recognition site for Alu I. This information was used to design a PCR based assay for simple and rapid mutation detection in DNA from blood samples to allow routine diagnosis of HCCAA. Studies at the protein level, allowed through E. coli expression of wildtype and L68Q mutated cystatin C genes, revealed that both protein variants effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation: 0.4 and 0.3 nM, respectively), but differ considerably in their tendency to dimerize and form aggregates. The initial dimerization of L68Q-cystatin C results in complete loss of biological activity and is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. This result might be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates. The three-dimensional structure of normal cystatin C, crystallized in a complex with cathepsin B, was elucidated by X-ray analysis and subsequent refinement of the structure to 3.0 A resolution. Besides pinpointing the cystatin C structures resulting in efficient target enzyme inhibition, the results demonstrated that the Leu-68 residue is buried in the hydrophobic core of the protein. Studies of the three-dimensional solution structure of wildtype cystatin C by NMR spectroscopy revealed that cystatin C dimers can be formed as a result of slight, localized structural changes under conditions preceding complete defolding and denaturation of the protein. Dimers of L68Q-cystatin C are likely similar but are formed at temperatures nearly 30 degrees C lower than needed for the wildtype protein, indicating that the Leu-68-Gln substitution lowers the transition temperature for unfolding. Thus, the results presented suggest that cystatin C provides a system where decreased stability of a mutant protein correlates with its amyloidogenic nature. The NMR results furthermore imply that the hydrophobic proteinase-binding region of cystatin C is directly involved in dimer formation and that compounds designed to interact with this region could serve as inhibitors to the dimerization, and likely also the subsequent amyloid formation process, of cystatin C in HCCAA patients.
Amino Acid Sequence↗
Characterization of a cysteine proteinase inhibitor induced during neuronal cell differentiation.
A rat homolog of human cystatin E/M was identified by differential display of transcripts induced during neuronal cell differentiation. A member of the family 2 cystatins, rat cystatin E/M is secreted, glycosylated and developmentally regulated. Rat cystatin E/M is expressed in brain, and is induced during differentiation of a conditionally immortalized E17 rat hippocampal cell line (H19-7) by bFGF or activated Raf via MEK-dependent and -independent signaling pathways. Rat cystatin E/M protein is increased post-transcriptionally in PC12 cells, and the protein is secreted into the medium of primary embryonal hippocampal cultures. Analysis of the K (i) of recombinant His-tagged rat cystatin E/M toward cathepsins B and H revealed that rat cystatin E/M has an inhibitor profile distinct from that of other members of the cystatin family. Motif swapping between rat cystatin E/M and human cystatin C, a well-characterized cystatin, identified some residues that can contribute to the specificity of inhibition. Taken together, these results describe a member of the cystatin family that has a distinct inhibitor profile and may play a role in neuronal development.
Animals↗
A cysteine protease inhibitor stored in the large granules of horseshoe crab hemocytes: purification, characterization, cDNA cloning and tissue localization.
A cysteine protease inhibitor with an apparent Mr = 12,600, designated limulus (L)-cystatin, was isolated from hemocyte lysates of the Japanese horseshoe crab (Tachypleus tridentatus), using two steps of chromatography, including dextran sulfate-agarose, and carboxymethylated papain-agarose. L-cystatin inhibits amidolytic activity of papain by forming a noncovalent 1:1 complex with an equilibrium constant (Ki) of 0.08 nM. It also inhibits cathepsin L (Ki = 0.17 nM) and ficin (Ki = 0.52 nM), but not argingipain (a bacterial cysteine protease) and calpains. A cDNA for L-cystatin was isolated and the open reading frame coded for a mature protein of 114 amino acids, of which 99 residues were confirmed by peptide sequencing. L-cystatin shows significant sequence identities to members of the family 2 cystatin, such as bovine colostrum cystatin (33%) and human cystatin S (31%). Northern blotting revealed expression of the mRNA in hemocytes and slightly in heart but expression was negligible in hepatopancreas, intestine, stomach, and muscle. Immunoblotting revealed the localization to be in the large granules of hemocytes. Furthermore, L-cystatin has an antimicrobial activity against Gram-negative bacteria, which is much stronger than that of chicken egg white cystatin. These data suggest that the large granule-derived L-cystatin serves synergistically to accomplish an effective defense against invading microbes, together with other defense molecules that are released in response to external stimuli.
Agglutinins↗
Cysteine proteinase inhibitors in human melanoma transplanted into nude mice.
Cysteine proteinase inhibitors in the human melanoma tissue transplanted into nude mice were found to increase in concentration during tumor growth. The activity (unit/g) at 8 weeks was about 3 times higher than the activity at 4 weeks after transplantation. The inhibitors were separated into two main forms (Mr about 76,000 and 10,000) with Sephacryl S-200 and/or Sephadex G-75 gel chromatography. The activities of the inhibitors of both molecular masses increased parallely during tumor growth. The high molecular mass inhibitor fractions reacted with antisera made against alpha-cysteine proteinase inhibitor (alpha-CPI, human kininogen) and against neutral low-molecular mass proteinase inhibitor (cystatin B). Free cystatin B appeared to be liberated in SDS-polyacrylamide gel electrophoresis following electroimmunoblotting with an antiserum to cystatin B. Similarly, free cystatin B was detected in gel chromatography on Sephadex G-75 after alkali treatment at pH 11.5. It may thus represent a cystatin B--cysteine proteinase complex mixed with alpha-CPI. The low molecular mass inhibitor fractions reacted with antisera made against cystatin A and cystatin B. When the low-molecular mass inhibitor fraction was subjected to isoelectric focusing, it was separated into three peaks with pIs 8.0, 7.4, and 6.0. The inhibitors with pI 8.0 and 7.4 reacted with antisera made against cystatin B, while the inhibitor with pI 6.0 reacted with antisera made against cystatin B and cystatin A.
Animals↗
Kidney function predicts the rate of bone loss in older individuals: the Cardiovascular Health Study.
BACKGROUND: Results of cross-sectional analyses of the association of kidney function with bone mineral density (BMD) have been conflicting. We examined the association of cystatin-C, a new marker of kidney function that is unrelated to lean mass, with initial and follow-up BMD, in an ancillary study of the Cardiovascular Health Study, a population-based cohort of individuals > or = 65 years old. METHODS: Two years after measurement of cystatin-C and other covariates, the first BMD was measured in Pittsburgh, Pennsylvania and Davis, California, by using dual energy x-ray absorptiometry. Follow-up BMD was measured in Pittsburgh 4 years later. Associations of cystatin-C with initial BMD and the change in BMD (%/y) at the hip were examined with linear regression. Analyses were conducted separately for men and women. RESULTS: In 1519 participants who had cystatin-C and initial BMD assessed, 614 had follow-up BMD. The percent annual change in BMD at the total hip by cystatin-C quartiles was -0.24, -0.13, -0.40, and -0.66%/y (first to fourth quartile) in women and -0.02, -0.30, -0.18, and -0.94%/y in men. After adjusting for potential confounders, cystatin-C was marginally associated with initial BMD in men but not women. Cystatin-C was associated with bone loss in men; after adjustment for weight loss, cystatin-C was not associated with bone loss in women. CONCLUSION: Kidney dysfunction, as assessed by cystatin-C, is associated with a more rapid loss of BMD at the hip, especially in men. Further studies are needed to confirm these findings and to determine whether this loss leads to an elevated risk of fracture.
Absorptiometry, Photon↗
Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids.
Six cysteine proteinase inhibitors were isolated from human urine by affinity chromatography on insolubilized carboxymethylpapain followed by ion-exchange chromatography and immunosorption. Physicochemical and immunochemical measurements identified one as cystatin A, one as cystatin B, one as cystatin C, one as cystatin S, and one as low molecular weight kininogen. The sixth inhibitor displayed immunochemical cross-reactivity with salivary cystatin S but had a different pI (6.85 versus 4.68) and a different (blocked) N-terminal amino acid. This inhibitor was tentatively designated cystatin SU. The isolated inhibitors accounted for nearly all of the cysteine proteinase inhibitory activity of the urinary pool used as starting material. The enzyme inhibitory properties of the inhibitors were investigated by measuring inhibition and rate constants for their interactions with papain and human cathepsin B. Antisera raised against the inhibitors were used in immunochemical determinations of their concentrations in several biological fluids. The combined enzyme kinetic and concentration data showed that several of the inhibitors have the capacity to play physiologically important roles as cysteine proteinase inhibitors in many biological fluids. Cystatin C had the highest molar concentration of the inhibitors in seminal plasma, cerebrospinal fluid, and milk; cystatin S in saliva and tears; and kininogen in blood plasma, synovial fluid, and amniotic fluid.
Body Fluids↗
Papain labelled with fluorescent thiol-specific reagents as a probe for characterization of interactions between cysteine proteinases and their protein inhibitors by competitive titrations.
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.
Animals↗
Decreased activity of cathepsins L + B and decreased invasive ability of PC3 prostate cancer cells.
Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L + B activity, and invasion through a Matrigel matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L + B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.
Cathepsin B↗
Expression of human salivary histatin and cystatin/histatin chimeric cDNAs in Escherichia coli.
We have previously constructed recombinants encoding the full-length and truncated forms of cystatin-SN and expressed these in the Escherichia coli expression system pGEX-2T, which expresses foreign sequences as fusion proteins with glutathione S-transferase (GST). Recombinant cystatins were produced and purified in large quantities. The full-length recombinant cystatin-SN exhibited comparable biological activity and secondary structure to natural cystatin, validating the use of the full-length and mutant recombinant proteins for structure-function studies of salivary molecules. In this study, we have expressed histatin-1 cDNA in the pGEX-3X vector and cystatin-SN/histatin-1 or cystatin-SN/histatin-3 chimeric cDNAs in the pGEX-2T vector. Gene splicing by overlap extension (SOE), a PCR-based method, was used for generating the chimeric cDNAs. Each construct was analyzed by DNA sequencing, which showed the correct junctions and reading frames between the GST/histatin-1 and the GST/cystatin/histatin cDNAs. Expression of histatin and cystatin/histatin chimeras was induced by IPTG and the production of the fusion proteins monitored by SDS-PAGE/Coomassie blue staining and in the case of the GST/cystatin/histatin fusion proteins, also by Western blot using anti-cystatin antibody. The results of these studies showed that we have successfully constructed recombinants encoding the individual and chimeric salivary molecules and efficiently expressed these in E. coli expression system pGEX. Purification and characterization of recombinant histatin and cystatin-histatin hybrid proteins are presently ongoing.
Amino Acid Sequence↗
Human cysteine proteinase inhibitors. Isolation, physiological importance, inhibitory mechanism, gene structure and relation to hereditary cerebral hemorrhage.
The isolation and characterization of six human cysteine proteinase inhibitors is reported. Their distribution in human biological fluids is also described and discussed with respect to physiological function. Studies on kininogen and cystatin C with respect to structure-function relationships and, as a result of the cystatin C studies, a general model for the mechanism of cysteine proteinase inhibition by cystatins are presented. The model was used for the construction of synthetic inhibitors which showed good inhibitory properties against papain and the streptococcal cysteine proteinase. Structures of cDNA and gene for normal human cystatin C are accounted for, as well as studies on the cystatin C gene in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). As a result of this an RFLP that showed total co-segregation with the disease was found. It was concluded that the disease is caused by a point mutation in the cystatin C structural gene and that the RFLP will be a most useful tool for diagnosis of HCCAA. The production of recombinant cystatin C in E. coli is also reported and its possible use for treatment of HCCAA is discussed.
Amino Acid Sequence↗
Serum neutrophil gelatinase-associated lipocalin as a marker of renal function in children with chronic kidney disease.
Very few biomarkers exist for monitoring chronic kidney disease (CKD). We have recently shown that serum neutrophil gelatinase-associated lipocalin (NGAL) represents a novel biomarker for early identification of acute kidney injury. In this study, we hypothesized that serum NGAL may also represent a biomarker for the quantitation of CKD. Forty-five children with CKD stages 2-4 were prospectively recruited for measurement of serum NGAL, serum cystatin C, glomerular filtration rate (GFR) by Ioversol clearance, and estimated GFR (eGFR) by Schwartz formula. Serum NGAL significantly correlated with cystatin C (r=0.74, P<0.000). Both NGAL and cystatin C significantly correlated with measured GFR (r=0.62, P<0.000; and r=0.71, P<0.000, respectively) as well as with eGFR (r=0.66, P<0.000 and r=0.59, P<0.000, respectively). At GFR levels of >or=30 ml/min per 1.73 m(2), serum NGAL, cystatin C, and eGFR were all significantly correlated with measured GFR. However, in subjects with lower GFRs (<30 ml/min per 1.73 m(2)), serum NGAL levels correlated best with measured GFR (r=0.62), followed by cystatin C (r=0.41). We conclude that (a) both serum NGAL and cystatin C may prove useful in the quantitation of CKD, and (b) by correlation analysis, NGAL outperforms cystatin C and eGFR at lower levels of measured GFR.
Acute-Phase Proteins↗