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PubMed2003

Decreased levels of cystatin C, an inhibitor of the elastolytic enzyme cysteine protease, in acute and subacute phases of kawasaki disease.

Elevation of tissue-destructive proteases has been reported in acute Kawasaki disease. Cystatin C is a naturally occurring inhibitor of elastolytic cysteine protease in humans. Serum cystatin C deficiency in human beings has been linked to atherosclerosis and aortic aneurysms. We investigated the serum levels of cystatin C during acute Kawasaki disease. Serum samples from 17 acute Kawasaki disease patients were collected before and after immunoglobulin therapy and also at a median of 17 days after the therapy. Eight adults and 10 children without intercurrent infections served as control patients. Children with Kawasaki disease prior to therapy had significantly lower levels of cystatin C compared to adults (p = 0.002) and control children (p = 0.001). The low levels persisted 1-106 days after the therapy. Compared to control children and adults, children with Kawasaki disease had significantly lower serum levels of cystatin C in the acute stage before immunoglobulin therapy and in the subacute phase after the immunoglobulin therapy.

Acute Disease↗
PubMed1994

The complete amino acid sequence of pituitary cystatin from chum salmon.

Cystatin, a cysteine proteinase inhibitor, was isolated from chum salmon (Oncorhynchus keta) pituitary glands by ion-exchange chromatography on Mono-Q, gel filtration on Superdex 75, and reverse-phase HPLC on an ODS following ethanol-ammonium acetate extraction. Salmon pituitary cystatin was equipotent to chicken egg-white cystatin in the papain inhibitory assay. The cystatin consists of 111 amino acid residues with two disulfide linkages formed between 66-75 and 89-109, and has 43% identical sequences with chicken egg-white cystatin with consensus sequences of reactive sites, Gly(4), Gln-X-Val-X-Gly (48-52), and Ile(Val)-Pro-Trp (96-98).

Amino Acid Sequence↗
PubMed2002

Molecular cloning and functional expression of cDNA encoding a cysteine proteinase inhibitor, cystatin, from Job's tears (Coix lacryma-jobi L. var. Ma-yuen Stapf).

A lambdaZAP II cDNA library was constructed from mRNA in immature seeds of the grass Job's tears. A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library. The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues. The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn. The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found. A remarkable characteristic of the peptide sequence of Job's-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice. The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The purified recombinant protein inhibited papain.

Amino Acid Sequence↗
PubMed2004

Identification of cysteine protease inhibitors that belong to cystatin family 1 in the cellular slime mold Dictyostelium discoideum.

Family 1 cystatins are cytosolic inhibitors of cysteine proteases, and they are conserved in higher eukaryotes. We characterized two newly identified family 1 cystatins of the cellular slime mold Dictyostelium discoideum, cystatin A1 and A2. Their recombinant proteins showed specific inhibitory activity against papain and cathepsin B, respectively. Using specific polyclonal antibodies, we found that cystatin A1 is stably expressed throughout the life cycle of Dictyostelium, whereas cystatin A2 expression is up-regulated during the course of development.

Amino Acid Sequence↗
PubMed2004

Studies on the interaction of papain with human placental cystatin by UV, fluorescence and CD spectroscopy.

The interaction of activated papain with low molecular weight cystatin (Mr 12500) purified from human placenta has been studied. Analysis of inhibition of caesinolytic activity of papain by cystatin showed stoichiometry of 1:1. Kinetic studies gave an inhibition constant (K(i)) value of 5.5 x 10(-8) M and association rate constant (K(+1)) value of 3.4 x 10(4) (M(-1) s(-1)). All spectroscopic studies showed conformational changes in both papain and cystatin on formation of complex. The data suggest perturbation of environment of aromatic residues and change of their native structure and conformation thereby shedding light on the behaviour of cystatins, especially interaction of placental cystatin with thiol protease inhibitors.

Circular Dichroism↗
PubMed2005

Role of cystatins in tumor neovascularization.

Cystatins form a large superfamily of proteins with diverse biologic activities. All members of the cystatin superfamily share the presence of one, two or three cystatin domains. Cystatins were initially believed to act mainly as inhibitors of lysosomal cysteine proteases. In recent years, however, there has been increased awareness of additional or alternate biologic functions for these proteins. In this review, the authors will discuss the most recent findings and hypotheses that suggest that some members of the cystatin superfamily may play important roles during tumor progression. Special emphasis is given to their potential role as novel anti-angiogenic agents.

Cystatins↗
PubMed2002

[Cres (cystatin-related epididymal spermatogenic) gene regulation and function].

The CRES (cystatin-related epididymal spermatogenic) protein defines a new subgroup in the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, unlike the ubiquitous expression of cystatin C, the Cres gene is preferentialy expressed in postmeiotic germ cells, the proximal caput epididymidis, and anterior pituitary gonadotrophs. Furthermore, CRES protein lacks two of the three consensus sites necessary for the cystatin inhibition of C1 cysteine proteases. Therefore, CRES may perform unique and tissue-specific functions in the reproductive and neuroendocrine systems. In the present review, we describe our studies on: 1. the Cres gene promoter and the transcriptional regulatory protein and their associated DNA binding sites that may be important for tissue-specific expression; and 2. the biochemical function of CRES protein. In brief, Northern blot, gel shift analyses, and transient transfection assays demonstrated that the C/EBP beta (CCAAT/enhancer binding protein) transcription factor is the predominant C/EBP family member expressed in the epididymis and gonadotroph cells and is necessary for high levels of Cres expression in these two tissues. In other studies, analyses of transgenic mice expressing a CAT reporter gene driven by 1.6 kb of Cres promoter revealed CAT mRNA and protein only in the germ cells. These studies suggest that the 1.6 kb of Cres 5' flanking sequence contains the required DNA elements for expression in the testis, but lacks the elements to correctly target expression of the reporter gene in the epididymis. Alternatively, repressor elements may be present. Finally, in vitro protease assays were performed to determine if CRES functions as a protease inhibitor. In contrast to cystain C, CRES did not inhibit the C1 cysteine protease papain but rather inhibited at nanomolar concentrations the serine protease PC2, a prohormone processing enzyme. Therefore, CRES is a new cross-class inhibitor that may regulate PC2 of PC2-like proteases and suggests a role for CRES in the regulation of prohormone and proprotein processing.

Amino Acid Sequence↗
PubMed1991

Steady-state cystatin mRNA levels in chicken tissues in response to estrogen.

A full length cDNA clone coding for the cysteine proteinase inhibitor, chicken cystatin, was used to hybridize to RNA extracted from various tissues of the hen during several well defined stages of the 25 h ovulatory cycle. Measurements were made immediately after egg-laying (ovulation), 6 h prior to ovulation (estrogen surge), and 16 h prior to ovulation (minimum estrogen levels). Cystatin was expressed in all tissues examined, being most abundant in lung, followed by brain, heart, oviduct, pectoral muscle, and liver in decreasing order. There were no significant differences in the cystatin mRNA levels at the three different time points for any of the tissues examined, and no differences in the 0.95 kb size of the message. Pharmacological doses of estradiol administered to immature female chickens decreased the steady-state cystatin mRNA levels as analyzed by Northern blots. There were no differences in the pattern of expression in the different tissues between rooster and hens. Our data suggest that estrogen has no direct effect on the regulation of cystatin expression in chicken tissues.

Animals↗
PubMed1991

Recombinant Q53E- and Q53N--chicken egg white cystatin variants inhibit papain, actinidin and cathepsin B.

A cloned synthetic gene coding for (AEF S1M M29I M89L) chicken egg white cystatin was modified site-specifically at position Q53 by cassette mutagenesis. Two recombinant variants were isolated from a pIN-III-ompA E. coli expression system and purified by Cm-papain affinity chromatography. The mutations at the position 53 were confirmed by amino acid composition and amino acid sequence analysis of the appropriate tryptic peptides. The complexes of both cystatin variants, the Q53E- and Q53N-variant with papain, display Ki values similar to those determined with native chicken cystatin. However, the Ki values of the complexes with actinidin are hundredfold and with cathepsin B three hundredfold higher than with the native chicken cystatin. The different inhibition kinetics of these variants compared to wild type chicken cystatin emphasizes the specificity of single amino acid substitutions for optimal contacts between the binding segments of enzyme and inhibitor.

Amino Acid Sequence↗
PubMed1990

Tissue distribution of an inducible cystatin in isoproterenol-treated rats.

A well-characterized, monospecific rabbit antiserum directed to an isoproterenol-inducible type 2 salivary cystatin was used for immunocytochemical localization of this cystatin in rat salivary glands, as well as in other organs of normal and isoproterenol-treated rats. Immunocytochemical analysis revealed a moderate staining of secretory granules within the acinar cells of submandibular glands, which was more pronounced in tissues obtained from female rats. In addition, the inducible cystatin was readily detected within granular convoluted tubule cells and striated duct cells of submandibular glands of both male and female rats, although not all such structures were stained. Cystatin was also localized in the proximal convoluted tubule cells of the kidney in isoproterenol-treated female rats. Western blotting and Ouchterlony double diffusion analysis showed that the cystatin from submandibular gland and kidney extracts was immunologically identical.

Animals↗
PubMed1993

Human cystatin D. cDNA cloning, characterization of the Escherichia coli expressed inhibitor, and identification of the native protein in saliva.

A cDNA coding for cystatin D, a human member of the cystatin protein family, has been cloned after specific amplification of reverse-transcribed parotid gland RNA. After replacing the segment encoding the putative 20-residue signal peptide with one encoding the Escherichia coli OmpA leader sequence, the cDNA was expressed in E. coli. The isolated recombinant protein exhibited Ki values of 1.2 nM and > 1 microM for papain and cathepsin B, respectively. An antiserum raised against recombinant cystatin D recognized a protein in human saliva with electrophoretical mobility identical to that of the recombinant protein. Immunoenzymatic analysis revealed that this cysteine proteinase inhibitor is present in human saliva and tears at concentrations of 3.8 and 0.5 mg/liter, respectively, while it was not detected in seminal plasma, blood plasma, milk, or cerebrospinal fluid. Cystatin D purified from human saliva by immunosorption displayed a heterogeneous N-terminal end, with sequences starting at residues 5, 7, 9, and 11 of the predicted N-terminal portion of the mature protein. On the basis of structural and functional properties, cystatin D represents a novel cysteine proteinase inhibitor possibly playing a protective role against proteinases present in the oral cavity.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of cystatin from duck egg white.

It is the second peptidase inhibitor, after ovostatin, which showing the same antipapain activity in egg white in different avian species implies differences in amino-acid sequences. Cystatin from duck egg white was purified by carboxymethylpapain affinity chromatography and size-exclusion HPLC. The purified inhibitor which showed partial identity in the immunodiffusion test with chicken egg white cystatin, had an apparent molecular mass of 9.3 kDa as determined by SDS/PAGE. IEF analysis revealed five molecular forms of pI in the range 7.8-8.4. The obtained cystatin was neither glycosylated nor phosphorylated as it is in the case of chicken cystatin. The determined Ki (0.005 +/- 0.001 nM) was similar to that reported for human and chicken cystatin C.

Animals↗
PubMed1999

Postnatal changes of gene expression for tissue inhibitors of metalloproteinase-1 and -2 and cystatins S and C, in rat submandibular gland demonstrated by quantitative reverse transcription-polymerase chain reaction.

The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.

Animals↗
PubMed1987

Inhibition of chicken calpain II by proteins of the cystatin superfamily and alpha 2-macroglobulin.

Inhibition of chicken calpain II by proteins of the cystatin superfamily and alpha 2-macroglobulin was investigated. Human liver cystatins A and B, human cystatin C, chicken cystatin and rat T-kininogen were found not to be inhibitory. Inhibition was, however, observed for bovine and rat kininogens, with Ki (inhibition constant) values of 0.8 nM and 30 nM respectively. alpha 2-Macroglobulin inhibits calpain with an initial rate constant of the order of 3 X 10(4) M-1.S-1. Calpain complexed with alpha 2-macroglobulin showed only limited reactivity towards azocasein, but reacted readily with the peptide substrate Suc-Leu-Tyr-4-methyl-7-coumarylamide and with L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64). The calpain in the complexes was at least partially protected from loss of activity due to autolysis. The calpain-alpha 2-macroglobulin complexes contained both the calpain subunits.

Animals↗
PubMed1984

Cystatin S: a cysteine proteinase inhibitor of human saliva.

An acidic protein of human saliva, which we named SAP-1 previously, is now shown to be an inhibitor of several cysteine proteinases. The protein inhibited papain and ficin strongly, and stem bromelain and bovine cathepsin C partially. However, it did not inhibit either porcine cathepsin B or clostripain. The mode of the inhibition of papain was found to be non-competitive. The name cystatin S has been proposed for this salivary protein in view of the similarities in activity and structure to other cysteine proteinase inhibitors such as chicken egg-white cystatin and human cystatins A, B, and C. The cystatin S antigen was detected immunohistochemically in the serous cells of human parotid and submaxillary glands.

Cystatin A↗
PubMed1991

Tight-binding inhibition of cathepsin S by cystatins.

Human cystatins A, B and C were purified, and their inhibition efficiency was tested with the cysteine proteinase cathepsin S. Cathepsin S was strongly inhibited by cystatins A and B in the subnanomolar range and by cystatin C in the picomolar range. Two steps of inhibition of cathepsin S by the cystatins which involve slow binding are discussed.

Amino Acid Sequence↗
PubMed1986

Human cysteine proteinases and their protein inhibitors stefins, cystatins and kininogens.

The cathepsins B, H and L of human origin were isolated in pure form in sufficient quantities for structural characterization. The complete amino acid sequence of human liver cathepsin B was determined. Partial amino acid sequences of the human kidney cathepsin H and L show the highly conserved region around the active site cysteine. The cysteine proteinase inhibitors stefin A, human stefin B and human cystatin C were isolated, characterized and sequenced. Their amino acid sequences are compared with sequences of other protein inhibitors of the stefin and cystatin family, showing a high degree of homology throughout both families. The stefin and cystatin family, together with newly discovered kininogen family belong to the same superfamily of cystatins. The constructed dendrogram shows that the most closely related inhibitors so far sequenced are human stefin B and rat liver TPI.

Amino Acid Sequence↗
PubMed1989

Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli.

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.

Base Sequence↗