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PubMedDate not supplied

Characterization of novel anti-cathepsin W antibodies and cellular distribution of cathepsin W in the gastrointestinal tract.

Human cathepsin W (lymphopain) is a cysteine protease that is restrictively expressed in cytotoxic cells, in particular NK cells. Several anti-cathepsin W monoclonal antibodies were tested with respect to their capability to detect cathepsin W by Western blot analysis and immunohistochemistry. Subsequently, the distribution of cathepsin W-expressing cells was studied in gastrointestinal tissue specimens using the antibody CW-401B1. All cathepsin W-positive cells had a 'lymphocyte phenotype'. Notably, samples from patients suffering from chronic inflammatory bowel disease (Crohn's disease, CD; ulcerative coliltis, UC) or autoimmune gastritis revealed variable amounts of cathepsin W-expressing cells. The relative portion of cathepsin W-positive cells among the infiltrating leukocytes (determined by CD45) differed remarkably. In autoimmune gastritis, cathepsin W-expressing cells made up for 65% of all CD45+ cells, whereas the corresponding values for CD and UC were 11% and 6%, respectively. These differences imply a distinct involvement of cytotoxic cells expressing cathepsin W in the pathogenesis among these diseases. Furthermore, it was tested whether the pro-inflammatory cytokines TNF-alpha and IFN-gamma can regulate cathepsin W gene expression in NK-92 cells. Both pro-inflammatory cytokines had only little effect on the cathepsin W gene expression of these cells.

Antibodies, Monoclonal↗
PubMed2003

Functional involvement of cathepsin W in the cytotoxic activity of NK-92 cells.

Human cathepsin W (lymphopain) is a papain-like cysteine protease of unknown function that is specifically expressed in natural killer (NK) cells and to a lesser extent in cytotoxic T cells (CTL). In order to analyze the functional importance of cathepsin W for the cytotoxic process, we investigated NK-92 cells that have an NK cell-like phenotype and express cathepsin W. NK-92 cells possess strong cytotoxic activity against Jurkat and K562 cells. The cytotoxic activity of NK-92 cells against K562 was decreased in the presence of antisense phosphorothioate oligonucleotides against the cathepsin W-cDNA. Western blot analysis showed that the impaired cytotoxic activity of NK-92 cells was accompanied by reduced amounts of cathepsin W in the antisense-treated cells. In addition, co-cultivation experiments between NK-92 and K562 cells revealed a time-dependent decrease of cathepsin W by Western blot and immunofluorescence analysis during the cytotoxic attack, whereas CD56 expression of NK-92 cells was not affected. During cytotoxic attack, cathepsin W was neither targeted to K562 cells or other subcellular compartments, as shown by immunofluorescence analysis. The decrease of cathepsin W protein was associated with stable cathepsin W transcript levels. Control experiments using HT-29 cells, which are resistant against NK-92-mediated cytotoxicity, showed no change of cathepsin W expression, implying that the decrease of cathepsin W in the NK-92/K562 assay is linked to the cytotoxic process. Although the exact function of cathepsin W with respect to its enzymatic activity and its site of action still needs to be elucidated, our data demonstrate for the first time that cathepsin W is important for cellular cytotoxicity mediated by NK cells.

Base Sequence↗
PubMed2001

Human cathepsin W, a cysteine protease predominantly expressed in NK cells, is mainly localized in the endoplasmic reticulum.

Human cathepsin W (also called lymphopain) is a recently described papain-like cysteine protease of unknown function whose gene expression was found to be restricted to cytotoxic cells. Here we demonstrate that cathepsin W is expressed predominantly in NK cells and, to a lesser extent, in CTLs. Quantitative RT-PCR revealed that NK cells contained approximately 21 times more cathepsin W transcript than CTLs. The predominant expression of cathepsin W in NK cells was further confirmed by Western blot analysis and immunohistochemistry. IL-2-mediated stimulation of NK cells and CTLs revealed a stronger up-regulation of the cathepsin W gene and protein expression in NK cells (7-fold) than in CTLs (2-fold). Transfection experiments of HeLa cells and biochemical analyses revealed that cathepsin W is exclusively "high mannose-type" glycosylated and is mainly targeted to the endoplasmic reticulum (ER). Interestingly, the ER localization of cathepsin W was also found in NK cells, in which colocalization studies revealed an overlapping staining of cathepsin W and Con A, an ER-specific lectin. Furthermore, subcellular fractionation of cathepsin W-expressing cells confirmed the ER localization and showed that cathepsin W is membrane associated. Based on the results of this study, cathepsin W might represent a putative component of the ER-resident proteolytic machinery. The constitutive expression in NK cells and the stronger up-regulation of cathepsin W by IL-2 in NK cells than CTLs suggest that cathepsin W is not just a marker of cytotoxic cells but is, rather, specifically expressed in NK cells.

Antibodies, Monoclonal↗
PubMed2000

Phylogenetic relationships and theoretical model of human cathepsin W (lymphopain), a cysteine proteinase from cytotoxic T lymphocytes.

The recently described cysteine proteinase cathepsin W, also known as lymphopain, which is expressed specifically by CD8+ T lymphocytes, is phylogenetically related to the cruzipain-like group of the C1 family of peptidases. We have constructed sequence alignments and a theoretical three dimensional homology model of cathepsin W. These have allowed the characterization of signature features of cathepsin W in particular and the cruzipain lineage in general. The signature features are (1) an extended loop structure, Gly 170-Trp 200, in the second or beta-sheet domain; (2) an additional disulfide bond, Cys 25/Cys 60; (3) an additional "orphan" cysteine, Cys 5; (4) an additional residue. Ala 11, inserted after the first beta-sheet sheet; and (5) an S2 pocket lined with Phe 68 and Phe 230 which explains the preference for substrates containing Leu at P2. Further, the model suggested that cathepsin W could exist as a dimer with the Cys 5 of each monomer forming a disulfide bond and the Arg 40 Phe 46 loop (RISFWDF) forming part of the dimeric interface. By comparing cathepsin W with other members of the cruzipain group and with other C1 peptidases, six conserved residues were identified which appear in general to be characteristic of the cruzipain group, and which differentiate cruzipain group members from other C1 peptidases including those of the related cathepsin L lineage. The signature residues of the cruzipain lineage are (cruzipain numbering) Asn 33, Trp 38, Ala 124, Leu 127, Leu 164, and Pro 174.

Amino Acid Sequence↗
PubMed2004

Identification of a novel isoform predominantly expressed in gastric tissue and a triple-base pair polymorphism of the cathepsin W gene.

In order to investigate the prevalence of potential polymorphisms of the cathepsin W gene, the complete cDNA of 50 dyspeptic patients was analyzed. From those 37 (74%) revealed the wildtype sequence, 6 samples (12%) contained independent single base pair changes including 4 silent and 2 with amino acid changes. Furthermore, a triple-base pair polymorphism was found in 7 samples (14%, 4x heterozygous, 3x homozygous) leading to the following changes: F(217)S, H(248)Y, and I(250)T. Furthermore, a novel alternative splice variant concerning intron 10 was identified in 6 samples (12%). Notably, this novel isoform was only found in samples of gastric mucosa lymphocytes, whereas peripheral NK cells expressed cathepsin W wildtype only. Taken together, this study demonstrated for the fist time that a genetic variant and a novel isoform of cathepsin W are present in about 14% and 12%, respectively, within the Caucasian population.

Alternative Splicing↗
PubMed1998

Genomic structure, chromosomal localization, and expression of human cathepsin W.

A 12 kb genomic fragment containing the entire open reading frame of the human cathepsin W was isolated and the genomic organization of this papain-like protease gene was determined. The 3.8 kb gene was mapped by fluorescence in situ hybridization to chromosome 11q13.1. The gene contained ten exons with introns ranging from 81 to 1119 bp. Four of the nine introns and a 5' untranslated exon were conserved when compared to related genes such as cathepsins L, K and S, whereas there was no similarity to the genomic organization of cathepsins B or C. In contrast to conserved splice site locations in other cysteine protease family members, the cathepsin W gene contained five unique locations. Furthermore, human cathepsin W cDNA was expressed, and was found to be localized within the rough endoplasmic reticulum in transiently transfected Cos-7 and Hela cells.

Cathepsin W↗
PubMed1997

Human cathepsin W, a putative cysteine protease predominantly expressed in CD8+ T-lymphocytes.

A 750-bp fragment of a novel human cysteine protease has been identified from the dbEST databank. PCR cloning and DNA sequencing yielded a 1.38-kb full-length cDNA which encodes a polypeptide of 376 amino acids. The protein consists of a putative 21-residue signal peptide, a 106-residue propeptide and a 252-residue mature protein. The deduced amino acid sequence contains the highly conserved residues of the catalytic triad of papain-like cysteine proteases: cysteine, histidine, and asparagine. Furthermore, the protein sequence possesses two potential N-glycosylation sites: one in the propeptide and one in the mature protein. Comparison of the amino acid sequence of human cathepsin W with other human thiol-dependent cathepsins revealed a relatively low degree of similarity (21-31%). In contrast to cathepsins L, S, K, B, H and O, cathepsin W contains a 21-amino acid peptide insertion between the putative active site histidine and asparagine residues and an 8-amino acid C-terminal extension. This unique sequence may indicate that cathepsin W belongs in a novel subgroup of papain-like proteases distinct from that of cathepsin L- and B-like proteases. Northern blot analysis indicates a specific expression of cathepsin W in lymphatic tissues. Further analysis revealed predominant levels of expression in T-lymphocytes, and more specifically in CD8+ cells. The expression of the protease in cytotoxic T-lymphocytes may suggest a specific function in the mechanism or regulation of T-cell cytolytic activity.

Amino Acid Sequence↗
PubMed2004

Characterization of murine cathepsin W and its role in cell-mediated cytotoxicity.

Cathepsin W is a member of the papain-like family of cysteine proteases. In this report, we have isolated the cDNA for murine CtsW (mCtsW) from a splenocyte library. The deduced 371-amino-acid sequence shares 68% identity with human CtsW and includes the conserved catalytic triad cysteine, histidine, and asparagine found in all members of this family. In addition to the fulllength form of mCtsW, we have isolated an alternatively spliced form of the mRNA that lacks a complete catalytic triad. An S1 nuclease protection assay and a Western blot analysis showed that mCtsW is mainly restricted to the CD8(+) T cell and natural killer cell compartments. In addition, we confirmed that, like its human homologue, mCtsW is localized mainly to the endoplasmic reticulum and its expression is up-regulated upon activation. We also characterized the mCtsW locus using bacterial artificial chromosome clones. The gene consists of 10 coding exons and 9 introns spanning 3.2 kb. To elucidate the physiologic role of this protease, we generated mice deficient in mCtsW. Our data establish that mCtsW is not required for cytotoxic lymphocyte-induced target cell death in vitro. In addition, mCtsW deficiency does not alter the susceptibility of cytotoxic lymphocytes to suicide or fratricide after degranulation. Thus, mCtsW does not have a unique role in target cell apoptosis or cytotoxic cell survival in vitro.

Alternative Splicing↗
PubMed1999

Molecular cloning and structural and functional characterization of human cathepsin F, a new cysteine proteinase of the papain family with a long propeptide domain.

A cDNA encoding a new cysteine proteinase belonging to the papain family and called cathepsin F has been cloned from a human prostate cDNA library. This cDNA encodes a polypeptide of 484 amino acids, with the same domain organization as other cysteine proteinases, including a hydrophobic signal sequence, a prodomain, and a catalytic region. However, this propeptide domain is unusually long and distinguishes cathepsin F from other proteinases of the papain family. Cathepsin F also shows all structural motifs characteristic of these proteinases, including the essential cysteine residue of the active site. Consistent with these structural features, cathepsin F produced in Escherichia coli as a fusion protein with glutathione S-transferase degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, a substrate commonly used for functional characterization of cysteine proteinases. Furthermore, this proteolytic activity is blocked by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cysteine proteinases. The gene encoding cathepsin F maps to chromosome 11q13, close to that encoding cathepsin W. Cathepsin F is widely expressed in human tissues, suggesting a role in normal protein catabolism. Northern blot analysis also revealed a significant level of expression in some cancer cell lines opening the possibility that this enzyme could be involved in degradative processes occurring during tumor progression.

Amino Acid Sequence↗
PubMed1999

Human cathepsins F and W: A new subgroup of cathepsins.

Human cathepsin F is a recently described papain-like cysteine protease of unknown function. To investigate the evolutionary relatedness to other human cathepsins, we determined the genomic organization and the chromosomal localization of cathepsin F and isolated its putative promoter region. The gene of human cathepsin F (CTSF) is composed of twelve exons and eleven introns and was found to be similar to that of cathepsin W but different from the cathepsins K, S, L, O, B, and C. The splice sites of nine out of the eleven introns were identical to those determined in the cathepsin W gene (CTSW), whereas introns one and ten were unique for CTSF. The 4. 7 kb gene was mapped to the long arm of chromosome 11 at position q13.1-3, a locus shared with CTSW. Phylogenetic analysis of human cathepsin protein sequences demonstrated that (i) cathepsins F and W are evolutionarily separated from other human cathepsins, and (ii) cysteine proteases closely related to human cathepsin W and F are also expressed in parasites and mammals. Based on these phylogenetic findings, on the presence of a particular protein motif ("ERFNAQ") in the propeptides of cathepsins F and W as well as the genomic organization and chromosomal localization of their genes, we concluded that F and W form a novel subgroup of cathepsin proteases. We suggest the naming "cathepsin F-like" proteases distinct from the previously described cathepsins "L- and B-like" subgroups.

Cathepsin F↗
PubMed2005

Upregulation of cathepsin W-expressing T cells is specific for autoimmune atrophic gastritis compared to other types of chronic gastritis.

AIM: To investigate a pathophysiological role of cathepsin W (CatW), a putative thiol-dependent cysteine protease, which is specifically expressed in cytotoxic lymphocytes, in different types of chronic inflammation of the gastric mucosa. METHODS: Gastric and duodenal biopsies of patients with Helicobacter pylori (H pylori)-associated active gastritis (Hp, n = 19), chemically induced reactive gastritis (CG, n = 17), autoimmune atrophic gastritis (AIG, n = 20), lymphocytic corpus gastritis (LG, n = 29), celiac disease (CD, n = 10), and corresponding controls (n = 24) were analyzed by immunohistochemistry for the expression of CatW and CD45. Furthermore, immunohistochemical double staining with anti-CD3 and anti-cathepsin was performed for the samples of AIG. RESULTS: Median values of CatW-expressing cells among CD45-positive immune cells were between 2% and 6% for normal gastric mucosa, CG, and LG, whereas the corresponding value was significantly increased for AIG (24.7%, P<0.001) and significantly decreased for HP (0.7%, P<0.05). Double staining with anti-CD3 and anti-CatW antibodies revealed that >90% of CatW-expressing cells in gastric mucosa of AIG were T cells. Duodenal mucosa had significantly more CatW/CD45-positive cells than normal gastric mucosa (median: 17.8% vs 2%, P<0.01). The corresponding proportion of CatW/CD45-postive cells was decreased in CD compared to duodenal mucosa (median: 2.1% vs 17.8%, P<0.05). CONCLUSION: The opposite findings regarding the presence of CatW-positive cells in AIG (increase) and CD (decrease) reflects the different cellular composition of immune cells involved in the pathogenesis of these diseases.

Autoimmune Diseases↗
PubMed1998

Human cathepsin F. Molecular cloning, functional expression, tissue localization, and enzymatic characterization.

A cDNA for a novel human papain-like cysteine protease, designated cathepsin F, has been cloned from a lambdagt10-skeletal muscle cDNA library. The nucleotide sequence encoded a polypeptide of 302 amino acids composed of an 88-residue propeptide and a 214-residue mature protein. Protein sequence comparisons revealed 58% homology with cathepsin W; about 42-43% with cathepsins L, K, S, H, and O; and 38% with cathepsin B. Sequence comparisons of the propeptides indicated that cathepsin F and cathepsin W may form a new cathepsin subgroup. Northern blot analysis showed high expression levels in heart, skeletal muscle, brain, testis, and ovary; moderate levels in prostate, placenta, liver, and colon; and no detectable expression in peripheral leukocytes and thymus. The precursor polypeptide of human recombinant cathepsin F, produced in Pichia pastoris, was processed to its active mature form autocatalytically or by incubation with pepsin. Mature cathepsin F was highly active with comparable specific activities toward synthetic substrates as reported for cathepsin L. The protease had a broad pH optimum between 5.2 and 6.8. Similar to cathepsin L, its pH stability at cytosolic pH (7.2) was short, with a half-life of approximately 2 min. This may suggest a function in an acidic cellular compartment. Transient expression of T7-tagged cathepsin F in COS-7 cells revealed a vesicular distribution of the gene product in the juxtanuclear region of the cells. However, contrary to all known cathepsins, the open reading frame of the cathepsin F cDNA did not encode a signal sequence, thus suggesting that the protease is targeted to the lysosomal compartment via an N-terminal signal peptide-independent lysosomal targeting pathway.

Amino Acid Sequence↗
PubMed2006

Microarray analysis of gene expression associated with extrapulmonary dissemination of tuberculosis.

OBJECTIVE: Although extrapulmonary organs are involved in 20% of patients with tuberculosis, the host genetic factors associated with the extrapulmonary dissemination of tuberculosis are not yet known. The aim of this study was to identify the host genetic factors associated with the extrapulmonary dissemination of tuberculosis by comparing gene expression profiles of patients who had recovered from extrapulmonary tuberculosis and those who had recovered from pulmonary tuberculosis. METHODS: Five patients from each group were enrolled. Total RNA was extracted from peripheral blood mononuclear cells that had been incubated for 48 h with whole lysate of Mycobacterium tuberculosis (H37Rv, 0.5 microg/mL). Gene expression profiles were acquired using the GeneChip array and its applied systems. Gene expression profiles from five patients with previous extrapulmonary tuberculosis and one pooled control sample from five patients with previous pulmonary tuberculosis were analysed and compared. Genes that were expressed concordantly in more than 80% of arrays and that showed more than twofold changes in at least one array among samples from patients who had recovered from extrapulmonary tuberculosis were identified. RESULTS: Compared with the control sample, the expression of 16 genes, including those for tumour necrosis factor (TNF)-alpha and cathepsin W, was increased, and the expression of 45 genes including that for TNF-receptor superfamily member 7 (TNFRSF7), was decreased in the extrapulmonary tuberculosis patients. The altered expression of the TNF-alpha, cathepsin W and TNFRSF7 genes was confirmed by quantitative RT-PCR. CONCLUSIONS: Altered expression of the genes for TNF-alpha, cathepsin W and TNFRSF7 may be risk factors for the extrapulmonary dissemination of tuberculosis in humans.

Adult↗
PubMed2000

Programming for cytotoxic effector function occurs concomitantly with CD4 extinction during CD8(+) T cell differentiation in the thymus.

CD4(+) T cells are generally specialized to function as helper cells and CD8(+) T cells are generally specialized to function as cytotoxic effector cells. To explain how such concordance is achieved between co-receptor expression and immune function, we considered two possibilities. In one case, immature CD4(+)CD8(+) thymocyte precursors might first down-regulate expression of one co-receptor molecule, with the remaining co-receptor molecule subsequently activating the appropriate helper or cytotoxic functional program. Alternatively, we considered that the same intrathymic signals that selectively extinguished expression of one or the other co-receptor molecule might simultaneously initiate the appropriate helper or cytotoxic functional program. In the present study, we attempted to distinguish between these alternatives by examining thymocyte precursors of CD8(+) T cells for expression of Cathepsin C and Cathepsin W, molecules important for cytotoxic effector function. We report in developing thymocytes that Cathepsin C and Cathepsin W are expressed coordinately with extinction of CD4 co-receptor expression. We conclude that CD4 extinction and initiation of the cytotoxic functional program occurs simultaneously during differentiation of CD8(+) T cells in the thymus.

Animals↗
PubMed2003

Constitutive expression of cytotoxic proteases and down-regulation of protease inhibitors in LGL leukemia.

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder often associated with rheumatoid arthritis. The etiology of LGL leukemia is not known. In order to better understand the pathogenesis of LGL leukemia, we analyzed differential gene expression using microarray technology. We found that approximately 80 genes were up-regulated and 12 genes were down-regulated when compared to normal peripheral blood mononuclear cells (PBMC). In the present study, we were interested in a group of genes involved in cytotoxic function. The up-regulated genes involved in cytotoxic function were serine proteinases (granzymes A, B, H and K) cysteine proteinases [cathepsin C, cathepsin W (lymphopain)], calpain small subunit and caspase-8. In addition, a pore-forming protein perforin, was also up-regulated. Northern blot analysis and RNase protection assays (RPA) confirmed that these genes were over-expressed in the majority of samples from LGL leukemia patients. Of interest, proteolytic inhibitors such as cystatin C, A, alpha-1 antitrypsin and metalloproteinase inhibitors were down-regulated in leukemic LGL when compared to normal peripheral blood mononuclear cells. Importantly, the pattern of gene expression in leukemic LGL resembles that seen in activated cytotoxic T cells (CTL).

Cathepsin C↗
PubMed2004

Cathepsins K, L, B, X and W are differentially expressed in normal and chronically inflamed gastric mucosa.

The expression of cathepsins K, L, B, X and W was studied by quantitative RT-PCR in normal and inflamed gastric mucosa (antrum, corpus, cardia). Cathepsins B, L, K and X were expressed ubiquitously. In contrast, cathepsin W was expressed at very low levels. Infection by Helicobacter pylori caused a significant induction of cathepsin X (p<0.008), whereas the other cathepsins were not or only locally affected by H. pylori infection or reflux disease. Immunohistochemistry revealed specific expression of cathepsin X (macrophages), cathepsin K (parietal cells) and cathepsin W (lymphocytes), whereas cathepsins B and L were predominantly expressed in epithelial cells.

Cathepsin B↗