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PubMed1992

Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin. Molecular cloning and expression studies.

A full-length cDNA clone for a cysteine proteinase inhibitor (cystatin) was isolated from a lambda gt10 cDNA library of immature corn kernels by screening with a mixture of cDNA inserts for oryzacystatins I and II. The cDNA clone spans 960 base pairs, encoding a 135-amino-acid protein containing a signal peptide fragment. The protein, named corn cystatin I, is considered to be a member of the cystatin superfamily, since it contains the commonly conserved Gln-Val-Val-Ala-Gly region that exists in most known cystatins as a probable binding site and is significantly similar to other cystatins in its overall amino acid sequence. Corn cystatin I expressed in Escherichia coli showed a strong papain-inhibitory activity. Northern blot analysis showed that the amount of mRNA for corn cystatin I reaches a maximum 2 weeks after flowering and then decreases gradually.

Amino Acid Sequence↗
PubMed1999

Cysteine protease inhibitory activity and levels of salivary cystatins in whole saliva of periodontally diseased patients.

The 3 human salivary cystatins S, SA and SN are multifunctional proteins that possess a cysteine protease inhibitory property, but their ability to act as such is very different (SN > SA >> S). One form, S, also appears to possess antibacterial properties towards the bacterium Porphyromonas gingivalis, often associated with periodontal diseases. In this study we measured the total cystatin inhibitory activity and the levels of each salivary cystatin in the whole saliva of 8 periodontally diseased patients and 2 groups of control subjects (n = 6 and n = 10). The total cystatin inhibitory activity and the total salivary cystatin concentration in the periodontally diseased patients were found to be lower than the controls (p < or = 0.005). The concentration of S was depleted to levels that would not allow it to be an effective antibacterial agent, and the concentration of SA, although depleted in some cases, was still present at sufficient levels to allow it to act as an effective physiological inhibitor of cathepsin L. The concentration of cystatin SN was also depleted in the periodontally diseased patients, but was still present in sufficient quantities to act as an effective physiological cysteine protease inhibitor of cathepsins H and L. In comparison, the concentration of all 3 salivary cystatins in the control subjects were sufficient to enable these proteins to be both effective physiological cysteine protease inhibitors and antibacterial agents.

Adult↗
PubMed2004

Unexpected intracellular localization of the AMD-associated cystatin C variant.

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.

Aging↗
PubMed2005

A virus essential for insect host-parasite interactions encodes cystatins.

Cotesia congregata is a parasitoid wasp that injects its eggs in the host caterpillar Manduca sexta. In this host-parasite interaction, successful parasitism is ensured by a third partner: a bracovirus. The relationship between parasitic wasps and bracoviruses constitutes one of the few known mutualisms between viruses and eukaryotes. The C. congregata bracovirus (CcBV) is injected at the same time as the wasp eggs in the host hemolymph. Expression of viral genes alters the caterpillar's immune defense responses and developmental program, resulting in the creation of a favorable environment for the survival and emergence of adult parasitoid wasps. Here, we describe the characterization of a CcBV multigene family which is highly expressed during parasitism and which encodes three proteins with homology to members of the cystatin superfamily. Cystatins are tightly binding, reversible inhibitors of cysteine proteases. Other cysteine protease inhibitors have been described for lepidopteran viruses; however, this is the first description of the presence of cystatins in a viral genome. The expression and purification of a recombinant form of one of the CcBV cystatins, cystatin 1, revealed that this viral cystatin is functional having potent inhibitory activity towards the cysteine proteases papain, human cathepsins L and B and Sarcophaga cathepsin B in assays in vitro. CcBV cystatins are, therefore, likely to play a role in host caterpillar physiological deregulation by inhibiting host target proteases in the course of the host-parasite interaction.

Amino Acid Sequence↗
PubMed2000

Reference ranges for plasma cystatin C and creatinine measurements in premature infants, neonates, and older children.

AIM: To establish a reference range in the paediatric population for the new glomerular filtration rate (GFR) marker, cystatin C, and to compare it with that of creatinine. METHODS: Cystatin C and creatinine were measured by particle enhanced nephelometric immunoassay (PENIA) and fixed interval Jaff¿e methods, respectively, in 291 children aged 1 day to 17 years, including 30 premature infants with gestational ages ranging from 24 to 36 weeks. RESULTS: In the premature infants, concentrations of both cystatin C and creatinine were significantly raised compared with term infants, with cystatin C concentrations being between 1.10 and 2.06 mg/litre and creatinine between 32 and 135 micromol/litre. In premature infants, there was no significant relation between gestational age and cystatin C or creatinine concentration. Creatinine concentrations fell to a nadir at 4 months of age, rising gradually to adult values by about 15-17 years of age, in contrast to cystatin C, which fell to a mean concentration of 0.80 mg/litre by the 1st year of life, and remained constant throughout adulthood up to the age of 50 years. Neither analyte showed any influence of sex. CONCLUSION: The measurement of cystatin C, rather than creatinine, is more practical for monitoring GFR changes in the paediatric population.

Adolescent↗
PubMed1993

Induction of type 2 salivary cystatin in immunological and chemical kidney injury.

We have previously reported that isoproterenol induces type 2 salivary cystatin in both submandibular glands and kidney tubule cells of rats but not in any other organs examined. In the present study, we investigated whether this salivary protein is induced in other conditions that show kidney tubule injury. Immunocytochemistry, using a monospecific antiserum to this cystatin, revealed specific staining within the proximal tubule epithelium of the cortex as well as in the inner and outer stripe of the medulla of immunologically and chemically injured rats. Cystatin could not be detected in kidneys from healthy rats by means of immunocytochemistry. Weak staining was found in 3/3 kidneys of rats treated with turpentine and in 5/5 animals treated with potassium dichromate. In rats treated with puromycin, cystatin could not be demonstrated in 5/5 animals having proteinuria of less than 100 mg/24 h; however, moderate staining was observed in 4/5 puromycin-treated rats having proteinuria greater than 100 mg/24 h. In Heymann nephritis, cystatin was present in 7/31 kidneys with proteinuria lasting 6 to 15 weeks and in none (0/7) with proteinuria of shorter duration. Strong staining was also observed in 10/10 kidneys from rats with moderate-to-severe chronic serum sickness. This study shows that elaboration of type 2 cystatin in rats is not limited to salivary glands and, with our previous study, suggests that induction of this cysteine inhibitor may represent a local response to generalized tissue injury in both submandibular and renal tissues. These findings further demonstrate that induction of cystatin in salivary glands is not unique to these glands and suggest that induction of this cysteine proteinase inhibitor may represent a local response to tissue injury caused by diverse mechanisms.

Animals↗
PubMed1993

[Effect of shampoo on human hair cystatin extraction].

It is known that cystatins have not only regulatory functions in cellular protein catabolism, but also other physiological functions against viral and/or bacterial infection. Recently we have fond cystatin in the human hair shaft. The activity of human hair cystatin in a water extract of pieces of it reached a plateau after 3-hour incubation. The activity of cystatin was compared among three different lengths of hair. The shorter the hair was cut, the more hair cystatin was extracted. Cystatin was more easily extracted by adding a water-soluble detergent. On the basis of these results, the loss of human hair cystatin from the hair shaft seemed to be affected by the condition of the hair surface and by the detergents in hair shampoo.

Cystatins↗
PubMed2004

Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem.

A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem. This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable K(i) values of 1.18 x 10(-10) M and 9.53 x 10(-11) M, respectively. The recombinant cystatins were found to be thermally stable up to 60 degrees C. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing.

Amino Acid Sequence↗
PubMed1998

Cystatin capture enzyme-linked immunosorbent assay for immunodiagnosis of human paragonimiasis and fascioliasis.

An ELISA was developed using chicken cystatin as a capture agent for the immunodiagnosis of paragonimiasis and fascioliasis. The assay detected specific antibodies to fluke cysteine proteinases without the need for purified proteinases. An ELISA plate was sensitized with chicken egg white cystatin, incubated with excretory-secretory (ES) products of adult flukes, and standard ELISA procedures were then followed. The ELISA plates incubated with the ES products of Paragonimus westermani and Fasciola sp. showed high reactivity to sera from patients with paragonimiasis westermani and fascioliasis, respectively. The capture ELISA showed little cross-reactivity with sera from patients with paragonimiasis and fascioliasis, which showed immunodiagnostic cross-reactivity in a conventional ELISA using crude fluke antigens. Moreover, the capture ELISA showed little reactivity with sera from patients with five other helminth diseases and from healthy volunteers. Omitting either sensitization with cystatin or incubation with fluke ES products abolished high ELISA reactivity, as did a prior exposure of the ES products to cystatin. The addition of papain to an incubation solution of the ES products greatly reduced ELISA reactivity. Incubating the cystatin-sensitized plates with partially purified cysteine proteinases from flukes instead of the ES products also maintained a similar high ELISA reactivity. These results indicate that the cystatin capture ELISA elicits a cystatin and fluke cysteine proteinase antigen-mediated reaction and measures fluke cysteine proteinase-specific antibodies. Prior exposure to low molecular weight inhibitors of cysteine proteinases and other proteinases, such as E-64, leupeptin, aprotinin, and pepstatin, had no effect, suggesting that these inhibitors can be added to cysteine proteinase preparations to prevent autoproteolysis. This assay has good sensitivity and high specificity and is useful for the immunodiagnosis of paragonimiasis and fascioliasis.

Animals↗
PubMed1989

Chicken egg white cystatin. Molecular cloning, nucleotide sequence, and tissue distribution.

A lambda gt11 chicken oviduct cDNA library was screened with a mixed synthetic oligonucleotide corresponding to amino acid residues 81-90 of chicken egg white cystatin, a cysteine proteinase inhibitor. Two initial cDNA clones of 367 and 431 bases were isolated. Both clones contained coding sequences for cystatin from amino acid residue 82 to the carboxyl end plus 3'-untranslated region and a poly(A)+ tail. The two clones utilized different polyadenylation signals located 55 nucleotides apart. Further screening of the library yielded a full-length cystatin cDNA. Sequence analysis indicated that cystatin contains an NH2-terminal extension of 23 amino acids which is probably a signal sequence. The cystatin cDNA hybridized to an mRNA of approximately 0.95 kilobase and was present in varying amounts in all chicken tissues examined. The highest concentration was found in the lung. Gizzard, brain, and heart contained lesser amounts of cystatin mRNA but considerably higher than oviduct. Among a limited number of embryonic tissues examined, significantly higher levels of the mRNA were found in liver and heart tissues when compared with the corresponding adult tissues. These results suggested that the expression of the chicken cystatin gene is tissue-dependent and under developmental control.

Amino Acid Sequence↗
PubMed1991

The effect of adrenergic agonists and antagonists on cysteine-proteinase inhibitor (cystatin) in rat saliva.

The effect of a number of adrenergic agonists and antagonists on the induction of rat salivary cystatin was investigated. A highly sensitive and specific radioimmunoassay was used to determine cystatin in rat whole saliva. Treatment for 10 consecutive days with a non-specific beta-adrenergic agonist isoproterenol, or the beta 1-adrenergic agonists dobutamine or methoxyphenamine, resulted in the induction of the salivary cystatin. Induction was also found in rats treated for 10 days with arterenol. Only trace quantities of cystatin could be detected in saliva of rats treated with the beta 2-adrenergic agonists terbutaline or salbutamol. When isoproterenol was injected concomitantly with the mixed beta-antagonist propranolol or the beta 1-adrenergic antagonists metaprolol, proctocol or atenolol the production of cystatin was totally suppressed. However, the beta 2-antagonist, ICI 118551, produced only a partial reduction in salivary cystatin induction elicited by isoproterenol. The findings suggest that the induction of salivary cystatin is regulated, in part, by beta 1-adrenergic receptor stimulation.

Adrenergic beta-Agonists↗
PubMed2005

Studies on Acanthocheilonema viteae cystatin: genomic organization, promoter studies and expression in Caenorhabditis elegans.

Cystatins are reversible, tightly binding inhibitors of cysteine proteases. Filarial cystatins have been ascribed immunomodulatory properties and have been implicated in protective immunity. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cystatin gene locus of A. viteae. The gene is composed of 4 exons separated by 3 introns and spans approximately 2 kb of genomic DNA. The upstream genomic sequence contains transcriptional factor binding sites such as AP-1 and NF-Y, an inverted CCAAT sequence, and a TATA box. To investigate sites of cystatin expression, Caenorhabditis elegans worms were transformed by microinjection with the putative promoter region and the first exon of the A. viteae cystatin gene fused to the reporter GFP. In transgenic worms fluorescence was observed in the pharyngeal and rectal gland cells suggesting that cystatin is secreted. Additionally, A. viteae cystatin was expressed in C. elegans to explore its potential as an expression system for filarial genes.

Journal Article↗
PubMed1998

Identification of cystatin B in human esophageal carcinoma, using differential displays in which the gene expression is related to lymph-node metastasis.

Identification of the genes that are specifically expressed in either tumor or normal tissue is important for understanding cancer biology. Using differential displays, we obtained one gene which was specifically expressed in normal tissue but is only rarely expressed in carcinoma tissue of the human esophagus. The sequence of this gene was identical with cystatin B, known to be one of the cysteine-proteinase inhibitors, mainly inhibiting cathepsin L. There is little information on the clinical significance of cystatin B expression in human esophageal carcinoma. We thus studied the mRNA expression of cystatin B in 45 tumor/normal pair specimens of the esophagus, using the semi-quantitative reverse transcriptase polymerase chain reaction technique. The expression of cystatin B in tumor tissue was found to be markedly decreased compared with that of the corresponding normal tissue. The cases with a tumor/normal ratio of less than 0.5 showed high frequency of lymph-node metastasis and more advanced clinical stage as compared with those whose tumor/normal ratio was equal to or more than 0.5. The decreased expression of cystatin B protein in carcinoma tissue was confirmed by immunohistochemical staining. Our study suggests that reduced expression of cystatin B in esophageal-carcinoma tissue is associated with lymph-node metastasis and may therefore prove to be a useful marker for predicting the biologic aggressiveness of human esophageal carcinoma.

Cathepsin L↗
PubMed2005

Cystatin C expression is associated with granule cell dispersion in epilepsy.

Human temporal lobe epilepsy (TLE) is associated with cellular alterations (eg, hilar cell death, neurogenesis, and granule cell dispersion) in the dentate gyrus but their underlying molecular mechanism are not known. We previously demonstrated increased expression of cystatin C, a protease inhibitor linked to both neurodegeneration and neurogenesis, during epileptogenesis in the rat hippocampus. Here, we investigated cystatin C expression in the dentate gyrus in chronic epilepsy and its association with neuronal loss and neurogenesis. In both rats with epilepsy and human patients with TLE, cystatin C expression was increased in glial cells in the molecular layer of the dentate gyrus, being most prominent in cases with granule cell dispersion. In patients with TLE, high cystatin C expression associated with greater numbers of polysialylated neural cell adhesion molecule-positive newborn cells in the molecular layer, although the overall number was decreased, indicating that the newborn cells migrate to abnormal locations in the epileptic dentate gyrus. These data thus demonstrate that cystatin C expression is altered during the chronic phase of epilepsy and suggest that cystatin C plays a role in network reorganization in the epileptic dentate gyrus, especially in granule cell dispersion and guidance of migrating newborn granule cells.

Adolescent↗
PubMed1995

Synthesis and secretion of procathepsin B and cystatin C by human bronchial epithelial cells in vitro: modulation of cathepsin B activity by neutrophil elastase.

Procathepsin B and cystatin C are found in human lung secretions. We investigated the capacity of human bronchial epithelial cells to synthesize and secrete these proteins. Immunoprecipitation of [35S]methionine-labeled proteins from cultured bronchial epithelial cell lysates, followed by denaturing gel electrophoresis and autoradiography, showed the presence of newly synthesized procathepsin B of M(r) 42,000; no mature form was detected. Cathepsin B in conditioned medium from epithelial cells was tagged with benzyloxycarbonyl-125I-tyrosyl-alanine-diazomethane before and after treatment of the medium with neutrophil elastase. Control medium again showed a predominant form of cathepsin B with a M(r) of 42,000, but upon treatment with neutrophil elastase this protein was converted to a M(r) of 38,000, similar to the active form previously found in lung secretions, and cathepsin B activity was generated. The medium also contained the cathepsin B inhibitor, cystatin C, but cystatins A, B, S, SN, SA, and kininogen were not detected. After removal of cystatin C from the medium, elastase was still required to activate procathepsin B. These results suggest that bronchial epithelial cells are a source of procathepsin B and cystatin C in lung secretions. Cleavage both of cystatin C and procathepsin B by neutrophil elastase is essential for the generation of cathepsin B activity in the medium.

Bronchi↗
PubMed2006

Estimating glomerular filtration rate in diabetes: a comparison of cystatin-C- and creatinine-based methods.

AIMS/HYPOTHESIS: We compared the predictive performance of a GFR based on serum cystatin C levels with commonly used creatinine-based methods in subjects with diabetes. SUBJECTS, MATERIALS AND METHODS: In a cross-sectional study of 251 consecutive clinic patients, the mean reference (plasma clearance of (99m)Tc-diethylene-triamine-penta-acetic acid) GFR (iGFR) was 88+/-2 ml min(-1) 1.73 m(-2). A regression equation describing the relationship between iGFR and 1/cystatin C levels was derived from a test population (n=125) to allow for the estimation of GFR by cystatin C (eGFR-cystatin C). The predictive performance of eGFR-cystatin C, the Modification of Diet in Renal Disease 4 variable formula (MDRD-4) and Cockcroft-Gault (C-G) formulas were then compared in a validation population (n=126). RESULTS: There was no difference in renal function (ml min(-1) 1.73 m(-2)) as measured by iGFR (89.2+/-3.0), eGFR-cystatin C (86.8+/-2.5), MDRD-4 (87.0+/-2.8) or C-G (92.3+/-3.5). All three estimates of renal function had similar precision and accuracy. CONCLUSIONS/INTERPRETATION: Estimates of GFR based solely on serum cystatin C levels had the same predictive potential when compared with the MDRD-4 and C-G formulas.

Adult↗
PubMed2000

Cystatin C--an accurate marker of glomerular filtration rate after renal transplantation?

The performance of serum cystatin C as a screening marker of reduced creatinine clearance in renal transplantation was evaluated and compared to serum creatinine. In addition we studied whether cystatin C accurately reflects creatinine clearance over the entire range of transplant function. Serum cystatin C, serum creatinine, and creatinine clearance were measured in 110 adult renal transplant recipients. Cystatin C detected reduced creatinine clearance with the high sensitivity of 95%. Serum cystatin C and serum creatinine did not differ regarding 90 and 95% sensitivity, derived from the receiver-operating characteristics plot. We demonstrated a strong correlation and linear association between 1/cystatin C and creatinine clearance over the entire range of transplant function, equivalent to that of 1/creatinine. In summary, serum cystatin C accurately reflects creatinine clearance over the entire range of transplant function and is as efficacious as serum creatinine to detect reduced creatinine clearance in renal transplant recipients.

Adult↗
PubMed1989

Amino acid sequence of an inducible cysteine proteinase inhibitor (cystatin) from submandibular glands of isoproterenol-treated rats.

We have previously reported the purification of an inducible cysteine proteinase inhibitor from submandibular glands of isoproterenol-treated rats by sequential gel filtration and ion-exchange chromatography [G. S. Bedi (1989) Arch. Biochem. Biophys. 270, 335-343]. This inhibitor is not detected in normal rat tissues but is induced in submandibular glands following beta-adrenergic stimulation of rats. In this study the complete amino acid sequence and the position of disulfide bridges of the purified protein were determined by automated Edman degradation of the protein and its tryptic, chymotryptic, and Staphylococcus aureus V8 protease peptides and were as follows: (sequence; see text) Computer analysis revealed the presence of 40-50% sequence identity between inducible cysteine proteinase inhibitor and cystatins from human saliva, human cystatin C, bovine cystatins, and chicken cystatins, all members of Family 2 cystatins. The inhibitor has little sequence similarity with rat liver and epidermal cysteine proteinase inhibitors, which belong to Family 1 cystatins.

Amino Acid Sequence↗