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At least 19 recordsLinked to original sources
PubMed1996

Isolation and characterization of the mouse cystatin B gene.

The cystatins make up a large superfamily of proteins that inhibit cysteine proteases. Recently, we showed that loss-of-function mutations in the human cystatin B gene are responsible for progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1). However, despite the known role of cystatin B in cysteine protease inhibition, it is not clear why decreased levels of this protein cause EPM1. To provide new insights into the biochemical and pathological mechanisms of EPM1, we are working toward developing an animal model for this disease. Here we present the mouse cystatin B nucleotide and amino acid sequence. We show that the mouse gene spans a 3-kb genomic region and contains 3 exons and 2 introns, identical to the structure of both the rat and human cystatin B genes. The amino acid sequence identity of the protein is 86%, 79%, and 71% to that of the rat, human, and bovine cystatin B proteins, respectively. In addition, we show that the mouse cystatin B gene is expressed in many tissues, similar to results observed previously in humans. Finally, we report the mapping of the mouse cystatin B gene (Stfb) to chromosome 10, further extending the synteny between this region of the mouse chromosome and human chromosome 21q22.3.

Amino Acid Sequence↗
PubMed1997

Novel cystatin B mutation and diagnostic PCR assay in an Unverricht-Lundborg progressive myoclonus epilepsy patient.

Two mutations in the cystatin B gene, a 3' splice mutation and a stop codon mutation, were previously found in patients with progressive myoclonus epilepsy of Unverricht-Lundborg type [Pennacchio et al. (1996): Science 271:1731-1734]. We present here a new mutation 2404deltaTC: a 2-bp deletion within the third exon of the cystatin B gene in an Unverricht-Lundborg patient. This mutation results in a frameshift and consequently premature termination of protein synthesis. Complete sequencing of the coding region and splice junctions of the cystatin B gene showed that neither of the two previously known mutations was present in this patient. The level of cystatin B mRNA in an immortalized cell line was found to be decreased, as had been reported for other Unverricht-Lundborg patients. The new mutation further supports the argument that defects in the cystatin B gene cause the Unverricht-Lundborg form of progressive myoclonus epilepsy. We describe a simple PCR method which can detect the 2404deltaTC deletion. This assay, together with previously described PCR assays for the other two known mutations, should prove useful in confirming clinically difficult diagnoses of Unverricht-Lundborg disease.

Amino Acid Sequence↗
PubMed1985

Amino acid sequence of the intracellular cysteine proteinase inhibitor cystatin B from human liver.

The complete amino acid sequence of the cysteine proteinase inhibitor cystatin B (formerly named CPI-B) from human liver was determined. The 98-residue sequence (Mr = 11,175) was obtained by automated solid-phase Edman degradation of a large cyanogen bromide fragment and peptides generated by enzymatic cleavage. The protein starts with a blocked Met-Met sequence which is presumably N-acetylated. The sequence shows that human cystatin B is a member of the family of intracellular cystatins; it is 79% identical with cystatin beta from rat liver, but contains only a single cysteine. Human cystatin B is able to form a dimer stabilized by noncovalent forces.

Amino Acid Sequence↗
PubMed1997

Identification of mutations in cystatin B, the gene responsible for the Unverricht-Lundborg type of progressive myoclonus epilepsy (EPM1).

Progressive myoclonus epilepsy (EPM1) is an autosomal recessive disorder, characterized by severe, stimulus-sensitive myoclonus and tonic-clonic seizures. The EPM1 locus was mapped to within 0.3 cM from PFKL in chromosome 21q22.3. The gene for the proteinase inhibitor cystatin B was recently localized in the EPM1 critical region, and mutations were identified in two EPM1 families. We have identified six nucleotide changes in the cystatin B gene of non-Finnish EPM1 families from northern Africa and Europe. The 426G-->C change in exon 1 results in a Gly4Arg substitution and is the first missense mutation described that is associated with EPM1. Molecular modeling predicts that this substitution severely affects the contact of cystatin B with papain. Mutations in the invariant AG dinucleotides of the acceptor sites of introns 1 and 2 probably result in abnormal splicing. A deletion of two nucleotides in exon 3 produces a frameshift and truncates the protein. Therefore, these four mutations are all predicted to impair the production of functional protein. These mutations were found in 7 of the 29 unrelated EPM1 patients analyzed, in homozygosity in 1, and in heterozygosity in the others. The remaining two sequence changes, 431G-->T and 2575A-->G, probably represent polymorphic variants. In addition, a tandem repeat in the 5' UTR (CCCCGCCCCGCG) is present two or three times in normal alleles. It is peculiar that in the majority of patients no mutations exist within the exons and splice sites of the cystatin B gene.

Amino Acid Sequence↗
PubMed1996

Mutations in the gene encoding cystatin B in progressive myoclonus epilepsy (EPM1)

Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is an autosomal recessive inherited form of epilepsy, previously linked to human chromosome 21q22.3. The gene encoding cystatin B was shown to be localized to this region, and levels of messenger RNA encoded by this gene were found to be decreased in cells from affected individuals. Two mutations, a 3' splice site mutation and a stop codon mutation, were identified in the gene encoding cystatin B in EPM1 patients but were not present in unaffected individuals. These results provide evidence that mutations in the gene encoding cystatin B are responsible for the primary defect in patients with EPM1.

Amino Acid Sequence↗
PubMed1997

Allelic heterogeneity of Mediterranean myoclonus and the cystatin B gene.

Mediterranean myoclonus is a progressive myoclonus epilepsy with autosomal recessive inheritance. Another form has been described in Finland, the so-called Baltic myoclonus. Mediterranean myoclonus and Baltic myoclonus are also known as Unverricht-Lundborg disease. Linkage analyses have shown that the genes for both these forms of myoclonus are closely linked to 21q22.3 DNA markers, suggesting that they are caused by mutations at the same locus (EPM1). Recently, two heterozygous mutations were found in the cystatin B gene in patients with Unverricht-Lundborg disease. We report recombinational and linkage disequilibrium mapping of EPM1, and cystatin B gene sequencing, in 14 consanguineous pedigrees with Mediterranean myoclonus. Linkage to 21q22.3 DNA markers was observed in all these families. Haplotype analysis suggests that a common mutation segregates within these pedigrees, and that this mutation is different from the common one responsible for the Finnish form of Unverricht-Lundborg disease. No mutation was found in the exons or splice junctions of the cystatin B gene in the 14 pedigrees.

Base Sequence↗
PubMed1997

Unstable insertion in the 5' flanking region of the cystatin B gene is the most common mutation in progressive myoclonus epilepsy type 1, EPM1.

Progressive myoclonus epilepsy type 1 (EPM1, also known as Unverricht-Lundborg disease) is an autosomal recessive disorder characterized by progressively worsening myoclonic jerks, frequent generalized tonic-clonic seizures, and a slowly progressive decline in cognition. Recently, two mutations in the cystatin B gene (also known as stefin B, STFB) mapping to 21q22.3 have been implicated in the EPM1 phenotype: a G-->C substitution in the last nucleotide of intron 1 that was predicted to cause a splicing defect in one family, and a C-->T substitution that would change an Arg codon (CGA) to a stop codon (TGA) at amino acid position 68, resulting in a truncated cystatin B protein in two other families. A fourth family showed undetectable amounts of STFB mRNA by northern blot analysis in an affected individual. We present haplotype and mutational analyses of our collection of 20 unrelated EPM1 patients and families from different ethnic groups. We identify four different mutations, the most common of which consists of an unstable approximately 600-900 bp insertion which is resistant to PCR amplification. This insertion maps to a 12-bp polymorphic tandem repeat located in the 5' flanking region of the STFB gene, in the region of the promoter. The size of the insertion varies between different EPM1 chromosomes sharing a common haplotype and a common origin, suggesting some level of meiotic instability over the course of many generations. This dynamic mutation, which appears distinct from conventional trinucleotide repeat expansions, may arise via a novel mechanism related to the instability of tandemly repeated sequences.

Base Sequence↗
PubMed1997

The NNP-1 gene (D21S2056E), which encodes a novel nuclear protein, maps in close proximity to the cystatin B gene within the EPM1 and APECED critical region on 21q22.3.

We have cloned and molecularly characterized a novel human cDNA that encodes a protein of about 52 kDa that was shown by immunocytochemistry to have a nuclear localization. Northern blot analysis of a variety of human tissues revealed that the gene for this novel nuclear protein, designated NNP-1 (HGMW-approved symbol D21S2056E), is ubiquitously expressed as a 2.0-kb transcript. The NNP-1 protein displays significant sequence similarity to the C47E12.7 protein of Caenorhabditis elegans (38% identity and 58% similarity) and the YD78 protein of Saccharomyces cerevisiae (36% identity and 54% similarity). The human NNP-1 gene was mapped to a 15-kb DNA interval on chromosome 21q22.3, close to markers D21S1459 and D21S1953, that is 25 kb distal to the gene encoding cystatin B. Finally, an as-yet unknown gene that is highly related to NNP-1 was found proximal to the gene encoding cystatin B, near marker D21S1458, which is approximately 75 kb centromeric to the NNP-1 gene.

Amino Acid Sequence↗
PubMed1997

Dodecamer repeat expansion in cystatin B gene in progressive myoclonus epilepsy.

Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1; MIM 254800) is an autosomal recessive disorder with onset between 6 and 13 years followed by variable progression to mental deterioration and cerebellar ataxia. It is a rare disorder but more common in Finland (1 in 20,000) and the western Mediterranean. Two point mutations in the cysteine proteinase inhibitor gene cystatin B (CSTB), proved that this gene is responsible for EPM1 (ref. 3). An extensive search in the CSTB gene revealed mutations accounting only for 14% of the 58 unrelated EPM1 alleles studied. Here we report that the majority of EPM1 alleles contain expansions of a dodecamer (12-mer) repeat located about 70 nucleotides upstream of the transcription start site nearest to the 5' end of the CSTB gene. Normal alleles contain 2 or 3 copies of this repeat whereas mutant alleles contain more than 60 such repeats and have reduced levels of CSTB messenger RNA in blood but not in cell lines. 'Premutation' CSTB alleles with 12-17 repeats show marked instability when transmitted to offspring.

Alleles↗
PubMed1988

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↗
PubMed1990

Evolution of proteins of the cystatin superfamily.

We have examined the amino acid sequences of a number of proteins that have been suggested to be related to chicken cystatin, a protein from chicken egg white that inhibits cysteine proteinases. On the basis of statistical analysis, the following proteins were found to be members of the cystatin superfamily: human cystatin A, rat cystatin A(alpha), human cystatin B, rat cystatin B(beta), rice cystatin, human cystatin C, ox colostrum cystatin, human cystatin S, human cystatin SA, human cystatin SN, chicken cystatin, puff adder cystatin, human kininogen, ox kininogen, rat kininogen, rat T-kininogens 1 and 2, human alpha 2HS-glycoprotein, and human histidine-rich glycoprotein. Fibronectin is shown not to be a member of this superfamily, and the c-Ha-ras oncogene protein p21 (Val-12) probably is not a member also. It was convenient to divide members of the superfamily into four types on the basis of the presence of one, two, or three copies of cystatin-like segments and the presence or absence of disulfide bonds. Evolutionary dendrograms were calculated by three methods, and from these we have constructed a scheme depicting the sequence of events in the evolution of these proteins. We suggest that about 1000 million years ago a precursor containing disulfide loops appeared, and that all disulfide-containing cystatins are derived from this. We follow the evolution of the proteins of the superfamily along four main lineages, with special attention to the part that duplication of segments has played in the development of the more complex molecules.

Amino Acid Sequence↗
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↗
PubMed1989

Cysteine proteinase inhibitors in human squamous cell carcinoma.

Cysteine proteinase inhibitors in Tris extract of human skin squamous cell carcinoma tissue were studied. From 17.2g of the tissue, approximately equal to 113.5 U inhibitor was obtained. With Sephadex G-75 gel chromatography, 3 papain inhibitor peaks of Mr approximately equal to 100,000, 38,000, and 12,000 were separated. In the immunodiffusion studies, Mr approximately equal to 100,000 fractions reacted with antisera made against human kininogen (alpha-cysteine proteinase inhibitor) and cystatin B. By using alkali treatment, free cystatin B was dissociated from the Mr approximately equal to 100,000 fractions. It is suggested that Mr approximately equal to 100,000 fractions contain cystatin B and alpha-cysteine proteinase inhibitor complexed with tissue proteinases. The Mr approximately equal to 38,000 fractions reacted with antiserum made against the papain inhibitor of Mr approximately equal to 43,000. The inhibitors in the Mr approximately equal to 12,000 fractions were identified as cystatin A and B on the basis of their reactions with antisera made against these inhibitors purified from human tissues.

Carcinoma, Squamous Cell↗
PubMed1989

Demonstration of proteinase inhibitors cystatin A, B and C in breast cancer and in cell lines MCF-7 and ZR-75-1.

Polyclonal rabbit antibodies to endogenous natural inhibitors of cysteine proteinases (cystatins) were used for their immunohistochemical demonstration in samples of breast cancers and in two breast cancer derived cell lines (MCF-7 and ZR-75-1). The influence of estrogen stimulation on the expression of these inhibitors was also studied. The results showed that all cystatins tested can be found equally inside carcinomatous cells or in surrounding connective tissue, as well as in both cell lines examined. Further, the expression of inhibitors can be regulated by estrogen, and a certain role of cystatins in the regulation of the mitotic activity and differentiation of mammary cells can be supposed.

Breast Neoplasms↗
PubMed1990

Cathepsin B and cystatins: evidence for a role in cancer progression.

The cysteine proteinase cathepsin B has been implicated in the progression of tumors from a premalignant to a malignant state. Activity of cathepsin B has been shown to be elevated in parallel with malignancy or metastatic potential of human and rodent tumors. These increases in cathepsin B activity correspond in part to increases in mRNA for cathepsin B and in part to reduced regulation by endogenous low Mr cysteine proteinase inhibitors. Most properties of tumor cathepsin B appear to be similar to those of cathepsin B from normal tissues. However, the subcellular distribution of cathepsin B is altered in tumors, resulting in association of cathepsin B with plasma membrane fractions or in release of high Mr forms of cathepsin B into the extracellular milieu. Since cathepsin B can degrade laminin, fibronectin and type IV collagen, we speculate that the presence of cathepsin B at the surface of tumor cells may contribute to the local dissolution of basement membrane observed during tumor cell extravasation. Direct evidence that cathepsin B plays a role in cancer progression awaits studies in which upregulation or downregulation of the expression of cathepsin B and its endogenous inhibitors is found to alter tumorigenesis, metastatic potential, etc.

Animals↗
PubMed1986

Detection of low molecular weight cysteine proteinase inhibitors by time-resolved fluoroimmunoassay.

A time-resolved fluoroimmunoassay was developed for the detection of 3 human low molecular weight cysteine proteinase inhibitors, ACPI (cystatin A), NCPI (cystatin B), and gamma-trace (cystatin C). Polystyrene tubes or polystyrene microtitration strips were used as solid phase. The rabbit anti-inhibitor immunoglobulins were used as the capture antibody, and, when labelled with europium, also as the detector antibody. The threshold sensitivity of the tests was 0.1 ng/ml for NCPI and 1 ng/ml for the others. All the 3 cysteine proteinase inhibitors, ACPI, NCPI, and gamma-trace, were detected in pooled serum samples of patients with kidney failure. gamma-Trace seemed to be quantitatively the major and ACPI the minor inhibitor. No other low molecular mass cysteine proteinase inhibitor was detected after isoelectric focusing of the 12 kDa area of gel filtered human serum.

Antibody Specificity↗
PubMed1997

Expression of cysteine protease inhibitors stefin A, stefin B, and cystatin C in human lung tumor tissue.

In human lung tumor tissue specimen (n = 73) concentrations of stefins A and B were found to be increased 2.0-fold (p < 0.01) and 1.3-fold (p < 0.01), respectively, as compared to matched normal tissue. Stefin A and B concentrations were higher in primary tumors than in secondary tumors, i.e. metastases from other organs to the lung (p < 0.01; p < 0.05, respectively). Cystatin C concentrations were rather low and did not differ between tumor and normal tissue. Both concentrations of stefins did not correlate with TNM stages. Stefin A was higher in squamous cell carcinoma than in adenocarcinoma (p < 0.01), while stefin B did not show such a difference. At investigation of a relationship between survival probability of patients with primary tumors it was found that increased stefin B concentrations and total cysteine-protease-inhibitory activities but not stefin A concentrations were positively correlated with survival probability. It is concluded that stefins A and B are major contributors to the cysteine protease inhibitory activity in primary lung tumors. Stefin B proved to be a prognostic factor, especially in squamous cell carcinoma.

Adolescent↗
PubMed1986

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↗