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PubMed1993

Induction of type 2 cystatin in rat submandibular glands by systemically administered agents.

An inducible type 2 cystatin has earlier been characterized in submandibular glands and kidneys of rats treated with isoproterenol, as well as in kidneys of rats with experimental renal disease. The purpose now was to determine whether giving agents that have systemic toxicity could also be associated with induction of cystatin in rat salivary glands. Female Wistar rats (200-250 g) were given isoproterenol, cyclocytidine, potassium dichromate or turpentine oil. After autopsy, the organs were sectioned, fixed in 10% formalin, and processed routinely. Paraffin sections were processed for both the peroxidase-antiperoxidase and the avidin-biotin-alkaline phosphatase immunocytochemical methods. The submandibular glands of rats given cyclocytidine had generalized, strong staining of acinar cells, as well as occasional weak staining within granular convoluted tubules. Animals given either potassium dichromate or turpentine oil exhibited moderate staining for cystatin in submandibular acini. Rats given isoproterenol as a positive control exhibited strong acinar staining throughout the submandibular gland, while the glands of untreated rats were unreactive. Inducible type 2 cystatin could not be detected in the parotid or sublingual glands, or in trachea, lung, stomach, small intestine, large intestine, spleen, liver and pancreas, after treatment with any of the systemic agents evaluated. The results indicate that elaboration of type 2 cystatin can be induced by a variety of systemically administered agents other than isoproterenol, and suggest that elaboration of type 2 cystatin may represent a more generalized response to tissue injury.

Ancitabine↗
PubMed1989

Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation.

A synthetic gene coding for the cysteine proteinase inhibitor (desSer1 Ile29 Leu89) chicken cystatin was cloned and expressed in E. coli. The gene was assembled from 12 oligonucleotides and inserted into vector pUC 8. Expression as fusion protein was performed in a temperature-inducible E. coli system. The expression product was synthesized as 20% of total E. coli protein. The fusion protein was purified, the chicken cystatin homologue was split off with CNBr and the N-terminal sequence confirmed up to position 37. The properties of the purified material correspond to those of natural chicken cystatin. The recombinant cystatin variant binds anti-chicken cystatin IgG, is inhibitorily active and displays Ki values with papain and with cathepsin B similar to those determined for natural chicken cystatin.

Amino Acid Sequence↗
PubMed1991

Identification of hematoxylin-stainable protein in epidermal keratohyalin granules as phosphorylated cystatin alpha by protein kinase C.

The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin alpha. However, only its pI value (4.7) differed from that of cystatin alpha (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [gamma-32P]ATP was incorporated into recombinant cystatin alpha by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin alpha and was inhibited significantly by H-7, a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin alpha. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin alpha and that the phosphorylation is catalyzed specifically by PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Antigenicity of cystatin-binding proteins from parasitic protozoan. Detection by a proteinase inhibitor based capture immunoassay (PINC-ELISA).

A novel immunoassay (PINC-ELISA) was designed using proteinase inhibitors of the cystatin superfamily (PINC) in the solid phase, to promote the selective capture of cysteine proteinases. The method was applied in the identification of papain-like antigens from parasitic protozoa. PINC of human origin, namely recombinant cystatin C (r-cystatin C) or low molecular weight kininogen were used in the assays to adsorb proteases contained in cell lysates from various trypanosomatids. The PINC-ELISA was at first optimized with the major cysteine proteinase from Trypanosoma cruzi (known as GP57/51 or cruzipain), an antigen whose serodiagnostic properties were previously established. Cruzipain is selectively adsorbed from crude extracts of T. cruzi onto PINC-coated wells; the finding that antibodies bind to epitopes located away from the sites of interaction with r-cystatin or low molecular weight kininogen has allowed for the screening of antibodies in chagasic sera, the methodology being advantageous in that it dispensed prior purification of the proteinase antigen. The PINC-ELISA was then carried out with lysates originating from Leishmania m. amazonensis (amastigotes) or Leishmania donovani (promastigotes). Complexes between solid-phase r-cystatin C and antigenic ligands in the lysates were again detected. The Leishmania molecules which bound to r-cystatin C, were respectively recognized by serum antibodies from mice chronically infected with L. amazonensis or from patients with visceral leishmaniasis. Direct evidence for the presence of cysteine proteinases in lysates from L. donovani was then obtained, using synthetic fluorogenic substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Salivary cystatins influence ingestion of capsaicin-containing diets in the rat.

Dietary capsaicin consumed by rats over several days induces cystatin-like substances in submandibular saliva. Yet the physiological role of these salivary proteins has not been thoroughly investigated. Salivary cystatins in the rat submandibular glands are known to be induced by chronic treatment with the sympathetic beta-agonist, isoproterenol. In the present study, the possible roles of the salivary proteins on food intake were examined by comparing consumption of a capsaicin-adulterated (0.05%) diet in rats with and without isoproterenol pretreatment (0.1 and 5.0 mg/kg, 5 days). Electrophoretic analysis performed prior to feeding trials revealed that the group pretreated with 5 mg/kg isoproterenol had large amounts of cystatin in the saliva compared with the group pretreated with 0.1 mg/kg isoproterenol and control group. The group treated with 5 mg/kg isoproterenol showed greater consumption of the capsaicin-adulterated diet than the other groups until the 3rd day of trials. Bilateral removal of the submandibular and sublingual glands neutralized the effects of isoproterenol. Induction of salivary cystatins by isoproterenol treatment was not mimicked by systemic and intragastric administration of capsaicin. These results suggest that cystatins are included in the salivary proteins induced by capsaicin and that they contribute to enhanced ingestion of the capsaicin diet. Induction of salivary cystatins may be triggered by irritation of the oral mucosa by capsaicin.

Analysis of Variance↗
PubMed2003

A new subgroup of the family 2 cystatins.

The cystatins are a superfamily of cysteine protease inhibitors. Several genes including Cres (cystatin-related epididymal spermatogenic), testatin, and cystatin T, have been identified that are related to the family 2 cystatins but lack critical consensus sites important for cysteine protease inhibition. In addition, these genes are primarily expressed in the reproductive tract suggesting they may have evolved to perform tissue-specific functions distinct from that of the typical cystatins. This review describes the CRES subgroup within the family 2 cystatins including potential new members and their putative functions in the reproductive tract.

Amino Acid Sequence↗
PubMed2003

Use of phage display to select novel cystatins specific for Acanthoscelides obtectus cysteine proteinases.

Cysteine proteinases from larvae of the common bean weevil, Acanthoscelides obtectus (Coleoptera: Bruchidae), were isolated by ion exchange affinity chromatography on a CM-Cellulose column and used to select mutant cystatins from a library made with the filamentous M13 phage display system. The library contained variant cystatins derived from the nematode Onchocerca volvulus cystatin through mutagenesis of loop 1, which contains the QVVAG motif that is involved in binding to proteinases. After three rounds of selection, the activity of variant cystatins against papain and cysteine proteinases from A. obtectus was assayed by ELISA. Two different variant cystatins (presenting amino acids DVVSA and NTSSA at positions 65-69) bound to A. obtectus cysteine proteinases more tightly than to papain. In contrast, the wild type had similar affinity for A. obtectus proteinases and for papain. These two selected variants cystatins have greater specificity towards A. obtectus cysteine proteinases than the original sequence and could represent good candidate genes for the production of transgenic plants resistant to this insect pest.

Animals↗
PubMed2001

Cloning and expression of cystatin, a potent cysteine protease inhibitor from the gut of Haemonchus contortus.

A cDNA encoding a cysteine protease inhibitor (cystatin) was identified by immunoscreening a Haemonchus contortus cDNA library with antisera from lambs vaccinated with a protective membrane protein complex (H-gal-GP) derived from the gut of the parasite. The cDNA sequence, designated Cys-1, showed significant levels of similarity with cystatins from several species of nematode as well as with human cystatin. Recombinant H. contortus cystatin was expressed in Escherichia coli in a soluble and functionally active form, which proved to be a potent inhibitor of both mammalian cathepsin B and native H. contortus cysteine proteases. Immunolocalization studies using antisera raised against recombinant H. contortus cystatin showed that the inhibitor was predominantly expressed in the cytoplasm of intestinal cells. To determine whether H. contortus had any protective capacity against infection, lambs were vaccinated with the recombinant molecule and subsequently given a single challenge infection. Although vaccination did not confer any protection against infection with H. contortus, as judged by faecal egg output or worm counts, cystatin will be a valuable tool in the analysis of the function of the cysteine proteases which are the subject of on-going study as potential vaccine components.

Amino Acid Sequence↗
PubMed1988

Interaction of the cysteine proteinase inhibitor chicken cystatin with papain.

The two forms of chicken cystatin, with different isoelectric points, that have been described previously were indistinguishable in analyses of amino- and carboxy-terminal residues, amino acid composition, and peptide maps. The two forms thus are highly similar and most likely differ only in an amide group or in a small charged substituent. The binding of either cystatin form to highly purified, active papain was accompanied by the same pronounced changes in near-ultraviolet circular dichroism, ultraviolet absorption, and fluorescence emission. These changes were compatible with perturbations of the environment of aromatic residues in one or both proteins of the complex, arising from local interactions or from a conformational change. Modification of the single tryptophan residue of cystatin, at position 104, with N-bromosuccinimide resulted in considerably smaller spectroscopic changes on binding of the inhibitor to papain, indicating that the environment of this residue is affected by the binding. Analogous modification of Trp-69 and Trp-177 of papain markedly affected the fluorescence changes observed on binding of cystatin to the enzyme, similarly suggesting that these two residues of papain are involved in the interaction. The fluorescence increase of papain at alkaline pH, arising from Trp-177 and due to deprotonization of the adjacent His-159, was abolished on binding of cystatin to the enzyme, further supporting the proposal that this region of papain participates in the interaction with the inhibitor. A stoichiometry of binding of either cystatin form to papain of 1:1 and a lower limit for the binding constant of 10(9) M-1 were determined by titrations monitored by either the ultraviolet absorption or fluorescence changes induced by the interaction.

Animals↗
PubMed1989

Kinetics of binding of chicken cystatin to papain.

The kinetics of binding of chicken cystatin to papain were studied by stopped-flow fluorometry under pseudo-first-order conditions, i.e., with an excess of inhibitor. All reactions showed first-order behavior, and the observed pseudo-first-order rate constant increased linearly with the cystatin concentration up to the highest concentration that could be studied, 35 microM. The analyses thus provided no evidence for a limiting rate resulting from a conformational change stabilizing an initial encounter complex, in contrast with previous studies of reactions between serine proteinases and their protein inhibitors. The second-order association rate constant for complex formation was 9.9 X 10(6) M-1 s-1 at 25 degrees C, pH 7.4, I = 0.15, for both forms of cystatin, 1 and 2. This value approaches that expected for a diffusion-controlled rate. The temperature dependence of the association rate constant gave an enthalpy of activation at 25 degrees C of 31.5 kJ mol-1 and an entropy of activation at 25 degrees C of -7 J K-1 mol-1, compatible with no appreciable conformational change during the reaction. The association rate constant was independent of pH between pH 6 and 8 but decreased at lower and higher pH in a manner consistent with involvement of an unprotonated acid group with a pKa of 4-4.5 and a protonated basic group with a pKa of 9-9.5 in the interaction. The association rate constant was unaffected by ionic strengths between 0.15 and 1.0 but decreased somewhat at lower ionic strengths. Incubation of the complex between cystatin 2 and papain with an excess of cystatin 1 resulted in slow displacement of cystatin 2 from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Cystatins↗
PubMed2004

Glycosylation modification improved the characteristics of recombinant chicken cystatin and its application on mackerel surimi.

The recombinant and glycosylation chicken cystatins were expressed and secreted in the broth of Pichia pastoris X-33 transformant with apparent molecular masses (M) of 14 and 55 kDa, respectively. The glycosylation cystatin (glycocystatin) contained a polysaccharide chain that was composed of 50 DP of mannose residues. Because of the polymannosyl chain, the inhibitory ability in glycocystatin was 90.8% of recombinant cystatin. In addition to freeze-thawing stability, the thermal and pH stabilities as well as the susceptibility of glycocystatin were also enhanced. Both cystatins could improve the mackerel surimi gel by inhibiting the gel softening, which was derived from the hydrolysis of catheptic cysteine proteinases. Despite the additional amount of glycocystatin (8 units), twice that of recombinant cystatin, the 40 and 15% increases in breaking force and deformation of gels were also observed. Accordingly, the surimi gel was further improved by enhancing the stability of chicken cystatin.

Animals↗
PubMed2005

Cystatins may confer viral resistance in plants by inhibition of a virus-induced cell death phenomenon in which cysteine proteinases are active: cloning and molecular characterization of a cDNA encoding cysteine-proteinase inhibitor (celostatin) from Celosia cristata (crested cock's comb).

Cystatins (cysteine proteinase inhibitors) have been recently used in plants as antiviral strategy against those viruses whose replication involves cysteine proteinase activity. We proposed an idea that cystatins may confer resistance by inhibition of a virus-induced cell-death phenomenon in which cysteine proteinases are active. To test this idea, a full-length cDNA library was constructed from the preflowering stage of Celosia cristata (crested cock's comb) leaves, and a cDNA clone with cystatin domain was isolated using an oligonucleotide probe designed on the basis of the conserved peptide of plant cystatins. It was expressed in an Escherichia coli expression system as a fusion protein. The purified recombinant product, termed 'celostatin' (Celosia cystatin), inhibited the enzymatic activity of papain indicating its cystatin activity and prevented TMV (tobacco mosaic virus)-induced hypersensitive-response cell death in Nicotiana glutinosa (a wild species of tobacco) leaves by 65-70% at the concentration of approx. 50 ng/ml. It also offered resistance against TMV and caused normal growth of the test plant. Since the activity of cysteine proteinases is not involved in the TMV replication process, we speculated that inhibition of the hypersensitive response by celostatin may be due to the inactivation of proteolysis involved in the plant cell death programme, a phenomenon that has already been reported in animal systems.

Amino Acid Sequence↗
PubMed1989

Interaction of chicken cystatin with inactivated papains.

Papain which was inactivated by covalent attachment of small substituents to the active-site cysteine, up to the size of a carbamoylmethyl group, bound with high affinity to chicken cystatin (Kd less than approximately 15 pM), although less tightly than did active papain (Kd approximately 60 fM). However, as the size of the substituent was increased further, the affinity decreased appreciably, generally in proportion to the size of the inactivating group. For instance the dissociation constants for papain inactivated with N-ethylmaleimide and [N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-amido-(4-guanido )butane were 0.17 and approximately 10 microM respectively. The spectroscopic changes accompanying the reaction of all but the most weakly binding (Kd greater than or equal to 2 microM) inactivated papains with cystatin were similar to those induced by the active enzyme. Interactions involving the reactive cysteine residue of papain are thus not crucial for high-affinity binding of the enzyme to cystatin, in accordance with a recently proposed model for the enzyme-inhibitor complex, based on computer docking experiments. In this model there is sufficient space around the reactive cysteine in the complex for a small inactivating group, explaining the tight binding of papains with such substituents. However, larger inactivating groups cannot be accommodated in this space and therefore must displace the inhibitor out of the tight fit with the enzyme, in agreement with the observed decrease in binding affinity with increasing size of bulkier substituents. The kinetics of binding of cystatin to inactivated papains were compatible with simple, reversible, bimolecular reactions, having association rate constants of (7-9) x 10(6) M-1 s-1 at pH 7.4, 25 degrees C, similar to what was shown previously for the binding of cystatin to active papain. The rate of association of the inhibitor with either active or inactivated papain thus appears to be primarily diffusion-controlled. The decreasing affinity of cystatin for papains inactivated with groups of increasing size was shown to be due to progressively higher dissociation rate constants, consistent with the greater impairment of fit between the binding regions of the two molecules.

Animals↗
PubMed1996

Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors.

A panel of intramolecularly quenched fluorogenic substrates containing the conserved QVVA and LVG inhibitory sequences of cystatin inhibitors was used to describe the specificity of the major cysteine proteinase of Trypanosoma cruzi (cruzipain or cruzain). This approach was based on the observations that: (1) cruzipain is strongly inhibited by chicken cystatin and rat T-kininogen, two representative members of cystatin families 2 and 3; (2) the QVVA- and LVG-containing substrates are specifically hydrolysed by papain-like proteinases; and (3) the cystatin-like motifs are similar to the proteolytically sensitive sequences in cruzipain that separate the pro-region and/or the C-terminal extension from the catalytic domain. Specificity constants (kcat/Km) were determined and compared with those of mammalian cathepsins B and L from rat liver lysosomes. Cruzipain and the mammalian proteinases cleaved cystatin-derived substrates at the same site, but their specificities differed significantly. Increased specificity for cruzipain was obtained by replacing amino acids at critical positions on both sides of the cleavage sites, especially at position P2'. The specificity constants (k(cat)/Km) obtained for the two substrates with a prolyl residue at P2' (O-aminobenzoyl-QVVAGP-ethylenediamine 2-4-dinitrophenyl and O-aminobenzoyl-VVGGP-ethylenediamine 2-4-dinitrophenyl) were about 50 times higher for cruzipain than for rat cathepsin L and about 100 times higher than for cathepsin B. Diazomethylketone derivatives, based on the non-prime sequence of cystatin-derived substrates, inhibited cruzipain irreversibly, but their inactivation rate constants were considerably lower than those for mammalian cathepsins B and L, confirming the importance of P' residues for cruzipain specificity.

Amino Acid Sequence↗
PubMed2002

Cystatins of filarial nematodes up-regulate the nitric oxide production of interferon-gamma-activated murine macrophages.

Cystatins of two filarial nematodes were studied with regard to their capacity to up-regulate the production of nitric oxide (NO) in vitro, and the effects were analysed. Recombinant cystatin of the human pathogenic filaria Onchocerca volvulus and of the rodent filaria Acanthocheilonema viteae significantly enhanced the NO production of interferon (IFN)-gamma-activated macrophages of BALB/c and C3H/HeJ mice. Truncated cystatins lacking the N-terminal protease inhibitory active site, and showing marginal protease inhibitory activity, up-regulated the NO production to the same extent as the full-length proteins, indicating that the effect on the NO production is independent of cysteine protease inhibition. NO did not contribute to the suppression of proliferative T cell responses exerted by filarial cystatins, as shown in other studies, since NO synthase inhibitors did not restore proliferative responses. The up-regulation of NO production induced by filarial cystatins was partly dependent on the production of interleukin-10 and tumour necrosis factor-alpha, since depletion of both cytokines by antibodies led to a diminution of the enhanced NO production by 22-48%. Our data suggest that filarial cystatins are potent triggers of the production of NO, a mediator which was shown to have a role as an effector molecule against filarial worms in vitro and in vivo.

Adjuvants, Immunologic↗
PubMed1992

Cystatin C levels in sera of patients with human immunodeficiency virus infection. A new avidin-biotin ELISA assay for its measurement.

A solid-phase enzyme-linked immunosorbent assay (ELISA) for determining human serum cystatin C is described. In 50 normal samples, cystatin C concentration was 1247 +/- 224 micrograms/L (mean +/- SD) which is in agreement with previously reported levels. Serum levels of cystatin C and beta 2-microglobulin (beta 2-M) were investigated in a time-course study during the development of human immunodeficiency virus (HIV) infection. We found a persistent and uniform increase in the beta 2-M concentration (2762 +/- 1239 micrograms/L). In contrast to beta 2-M, on the basis of cystatin C levels, we found two distinct populations, one of which demonstrated an increased concentration (1620 +/- 618 micrograms/L). Interestingly a second group (21% of patients) exhibited an initial significant decrease in cystatin C concentration with a mean value of 377 (range 55-850) micrograms/L, followed by an increase. The biphasic pattern of cystatin C serum, a major cysteine proteinase inhibitor, during the course of HIV infection suggests a possible role for these proteinases (or proteinase inhibitors) in the development of this syndrome.

Adolescent↗
PubMed2002

Expression of type 2 cystatin genes CST1-CST5 in adult human tissues and the developing submandibular gland.

Type 2 cystatins comprise a class of cysteine peptidase inhibitor presumed to mediate protective functions at various locations, including the oral cavity. Seven cystatin genes are clustered within a 300-kb region of human 20p11.2. "Salivary" cystatins, encoded by CST1, 2, 4, and 5, are present in saliva at significant levels but have also been reported in other secretions, such as tears, suggesting that during their evolution, these genes have acquired mechanisms directing differential tissue-specific expression. However, their patterns of expression, which might also provide additional clues to their individual functions, have not been determined. Gene-specific RNase protection assays were used to examine the qualitative and quantitative distribution of expression of these seven genes within a collection of 23 adult human tissues. The CST3 gene, encoding cystatin C, was expressed at modest levels in all tissues examined. The presumptive pseudogenes CSTP1 and CSTP2 were not expressed at detectable levels in any tissue. The CST1, 2, 4, and 5 genes were expressed in differential, tissue-specific patterns. Expression of CST2 and CST5 was restricted to the submandibular and parotid glands, while CST1 and CST4 were expressed in these tissues and in the lacrimal gland. Immunohistochemistry studies localized expression to the serous-type secretory end pieces. Coexpression of CST1 and CST4 was also observed in the epithelial lining of the gallbladder and seminal vesicle. The CST1 product was detected in the tracheal glands and CST4 in the kidney and prostate. Despite their different adult patterns of expression, analysis of CST1, 2, 4, and 5 mRNA levels in infant submandibular glands demonstrated a coordinate upregulation of expression of between 3.5 and 9 months of age. The patterns of cystatin gene expression are consistent with several proposed oral functions of the salivary cystatins but also suggest they are important in other locations and that, despite their close sequence similarity, they are individually specialized.

Adolescent↗
PubMed2002

Immunohistochemical localization of cathepsin L and cystatin A in normal skin and skin tumors.

Cathepsin L, a cysteine proteinase, and cystatin A, an inhibitor of cysteine proteinases, are thought to regulate the invasion and metastasis of malignant cells. In this study, the expression of cathepsin L and cystatin A in skin tumors was investigated immunohistochemically in order to examine the relationship between these two enzymes in the pathophysiology of malignant cells. Formalin-fixed and paraffin embedded specimens from normal skin, seborrheic keratoses, and squamous cell carcinomas were reacted with polyclonal antibodies against rat cathepsin L or cystatin a which cross-react to human cathepsin L and cystatin A, respectively. The consequent immunostaining of these enzymes was observed to be strong in normal skin (4 cases) and seborrheic keratosis (6 cases). In well-differentiated squamous cell carcinoma (SCC) (9 cases), staining for cathepsin L and cystatin A was moderately positive in differentiated tumor cells and negative in undifferentiated SCC (5 cases). The degree of staining of these enzymes was inversely correlated with the differentiation of the malignant cells. These results suggest that the immunohistochemical analysis of cathepsin L and cystatin A is a useful indicator for an aspect of malignancy in human epidermal keratinocytes.

Biomarkers, Tumor↗