Sequence analysis, tissue distribution, and expression of rat cathepsin S.

Cysteine proteases are involved in many diverse cellular processes ranging from processing of precursor proteins to intracellular degradation. In an effort to identify novel cysteine proteases, we used the polymerase chain reaction and primers directed against the catalytic sites of previously cloned cysteine proteases. From rat brain mRNA, a 600-base pair band was amplified; cloning and partial sequence analysis of this band resulted in the identification of cathepsins B and L and five novel sequences. The novel cDNAs contained a number of residues conserved in lysosomal cysteine proteases, including the active site residue His159 (papain numbering). In addition, the amino acid homology between the novel sequences and either cathepsins B, L, or H, ranged from 63 to 32%. The insert with highest homology was used to screen a rat brain cDNA library; a 1334-base pair cDNA was isolated and the nucleotide sequence determined. This sequence encodes an open reading frame of 330 amino acids which is 82% homologous to human cathepsin S, suggesting that this sequence represents rat cathepsin S. Northern blot analysis for rat cathepsin S revealed tissue-specific expression distinct from the distribution of cathepsin B and L. The regulation of expression of rat cathepsin S mRNA in response to thyroid-stimulating hormone was studied in a rat thyroid cell line FRTL-5. The level of cathepsin S mRNA was substantially increased in response to thyroid-stimulating hormone, whereas cathepsin B and cathepsin L mRNA levels were not altered by this treatment. A portion of cDNA encoding the predicted mature protein of rat cathepsin S was expressed as a glutathione S-transferase-fusion protein. The affinity-purified protein exhibited proteolytic activity with properties similar to bovine cathepsin S. Taken together, these results imply highly specific functions for cathepsin S.     

Cathepsin expression during skeletal development.

Cysteine proteinases, cathepsins B, H, K, L and S, have been implicated in several proteolytic processes during development, growth, remodeling and aging, as well as in a variety of pathological processes. For systematic analysis of cathepsin gene expression we have produced cDNA clones for mouse and human cysteine cathepsins. Northern analysis of a panel of total RNAs isolated from 16-19 different human and mouse tissues revealed the presence of mRNAs for cathepsin B, H, K, L and S in most tissues, but each with a distinct profile. Of the different cathepsin mRNAs, those for cathepsin K were clearly the highest in bone and cartilage. However, relatively high mRNA levels for the other cathepsins were also present in these tissues. To better understand the roles of different cathepsins during endochondral ossification in mouse long bones, cathepsin mRNAs were localized by in situ hybridization. Cathepsin K mRNAs were predominantly seen in multinucleated chondroclastic and osteoclastic cells at the osteochondral junction and on the surface of bone spicules. The other cathepsin mRNAs were also seen in osteoclasts, and in hypertrophic and proliferating chondrocytes. These observations were confirmed by immunohistochemistry and suggest that all cysteine cathepsins are involved in matrix degradation during endochondral ossification.     

Unique cathepsin D-type proteases in rat thoracic duct lymphocytes and in rat lymphoid tissues.

Two unique cathepsin D-type proteases apparently present only in rat thoracic duct lymphocytes and in rat lymphoid tissues are described. One, termed H enzyme, has an apparent molecular weight of similar to95,000; the other, termed L enzyme, has an apparent molecular weight of similar to45,000, in common with that of most cathepsins D from other tissues and species. Both enzymes differ from cathepsin D, however, by a considerably greater sensitivity to inhibition by pepstatin and by a smaller degree of inhibition by an antiserum which inhibits rat liver cathepsin D. H enzyme is converted to L enzyme by treatment with beta-mercaptoethanol; the relationship between the two enzymes remains unknown. H and L enzyme have been detected in rat lymphoid tissues and in mouse spleen, but they are not present in other rat tissues (liver, kidney, adrenals), rabbit tissues, calf thymus, bovine spleen, or human tonsils. As measured on acid-denatured bovine hemoglobin as substrate, both enzymes have pH activity curves identical with that of rat liver cathepsin D, with optimal activity at pH 3.6. Activity on human serum albumin is much less and also shows an optimum at pH 3.6; hence, neither enzyme has the properties of cathepsin E. Thiol-reactive inhibitiors have no effect on the activity of H and L enzyme; thus they do not belong to the B group of cathepsins. Additional information, discussed in this paper, leads us to conclude that partially purified H and L enzymes are cathepsin D-type proteases.     

Distribution of cathepsins B and H in rat tissues and peripheral blood cells

The concentrations of cathepsins B and H in various tissues and peripheral blood cells of rats were determined by means of sensitive immunoassays. The minimum detectable amounts of cathepsins B and H were 30 pg and 20 pg/assay, respectively, and the presence of endogenous thiol proteinase inhibitors did not interfere with the immunoassays. Cathepsin B was found at high levels in the kidney, vagina, spleen, and adrenal gland, and cathepsin H at high levels in the kidney, vagina, liver, lung, and spleen. Low levels of cathepsins B and H were present in the heart, skeletal muscle, and testis. The ratios of cathepsins B and H in various organs were different: the brain and adrenal gland contained much higher levels of cathepsin B than of cathepsin H, whereas the lung and liver contained higher levels of cathepsin H than of cathepsin B. In several organs such as the kidney, spleen, liver, and lungs, the level of cathepsins B plus H was much higher than that of thiol proteinase inhibitors (TPI-alpha + TPI-beta), whereas in tissues containing large amounts of TPI-alpha, such as the skin, esophagus and stomach, the level of inhibitors was higher than that of cathepsins B plus H. Of the peripheral blood cells tested, macrophages had the highest contents of cathepsins B and H, and so their level of cathepsins B plus H was much higher than that of TPI-alpha plus TPI-beta, whereas lymphocytes and neutrophils contained comparable amounts of proteinases and inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in cathepsin B mRNA levels in rat tissues suggest specialized functions

The tissue distribution of mRNAs encoding two lysosomal proteases, cathepsin B and cathepsin D, was examined using cloned cDNAs to probe Northern and dot blots of RNAs extracted from various rat tissues. Cathepsin B mRNA showed a wide range of variation in expression in the tissues analyzed with the highest concentrations found in spleen and kidney, while the cathepsin D mRNA levels were relatively uniform in these same tissues. Significant quantities of cathepsin B mRNA were detected in total RNA from isolated islets of Langerhans but was not detectable in equivalent amounts of RNA from whole pancreas. The wide variations in tissue levels of cathepsin B mRNA suggest that tissue specific controls may regulate its expression and are compatible with the participation of this protease in specialized cellular functions other than intralysosomal protein degradation.     

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: molecular cloning and gene expression during vitellogenesis and oocyte maturation

The cysteine proteinases cathepsins B and L are members of the multigene family of lysosomal proteases that have been implicated in the processing of yolk proteins (YPs) in teleost oocytes. However, the full identification of the type of cathepsins expressed in fish ovarian follicles and embryos, as well as their regulatory mechanisms and specific function(s), are not yet elucidated. In this study, cDNAs encoding cathepsins B, L, F, K, S, Z, C, and H have been isolated from the teleost Fundulus heteroclitus, and the analysis of their deduced amino acid sequences revealed highly similar structural features to vertebrate orthologs, and confirmed in this species the existence of cathepsin L-like, cathepsin B-like, and cathepsin F-like subfamilies of cysteine proteinases. While all identified cathepsins were expressed in ovarian follicles, the corresponding mRNAs showed different temporal expression patterns. Thus, similar mRNA levels of cathepsins L, F, S, B, C, and Z were found throughout the oocyte growth or vitellogenesis period, whereas those for cathepsin H and K appeared to decrease as vitellogenesis advanced. During oocyte maturation, a transient accumulation of cathepsins L, S, H, and F mRNAs, approximately a 3-, 1.5-, 1.6-, and 6-fold increase, respectively, was detected in ovarian follicles within the 20-25 hr after hormone stimulation, coincident with the maximum proteolysis of the oocyte major YPs. The specific temporal pattern of expression of these genes may indicate a potential role of cathepsin L-like and cathepsin F proteases in the YP processing events occurring during fish oocyte maturation and/or early embryogenesis.     

The heterogeneity of rat kidney lysosomes revealed by immunoelectron microscopic staining for cathepsins B and H

Summary The heterogeneity of lysosomes was studied by analyzing the immunostaining behavior of cathepsins B and H in rat kidney proximal tubule cells. Rat kidneys were fixed by perfusion and embedded in Lowicryl K4M. A protein A-gold technique was applied to serial sections and a double labeling technique to conventional sections. By analyzing the immunostaining behavior of cathepsins B and H in the same lysosomes which were cut into separate sections, four types of lysosomes were found: Type 1 positive for both proteinases; type 2 strongly positive for cathepsin B, but weakly or negative for cathepsin H; type 3 strongly positive for cathepsin H, but weakly or negative for cathepsin B; and type 4 negative for both proteinases. The double labeling by two different sizes of the protein A-gold probes showed these four types of lysosomes. The results indicate that there exists the lysosomal heterogeneity of the proteinase content in the kidney proximal tubule cells.     

The heterogeneity of rat kidney lysosomes revealed by immunoelectron microscopic staining for cathepsins B and H

The heterogeneity of lysosomes was studied by analyzing the immunostaining behavior of cathepsins B and H in rat kidney proximal tubule cells. Rat kidneys were fixed by perfusion and embedded in Lowicryl K4M. A protein A-gold technique was applied to serial sections and a double labeling technique to conventional sections. By analyzing the immunostaining behavior of cathepsins B and H in the same lysosomes which were cut into separate sections, four types of lysosomes were found: Type 1 positive for both proteinases; type 2 strongly positive for cathepsin B, but weakly or negative for cathepsin H; type 3 strongly positive for cathepsin H, but weakly or negative for cathepsin B; and type 4 negative for both proteinases. The double labeling by two different sizes of the protein A-gold probes showed these four types of lysosomes. The results indicate that there exists the lysosomal heterogeneity of the proteinase content in the kidney proximal tubule cells.     

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.     

Cellular retinoic acid-binding protein messenger RNA: levels in rat tissues and localization in rat testis.

Studies were conducted to explore the tissue- and cell-specific regulation of cellular retinoic acid-binding protein (CRABP) expression in the rat. Two studies were carried out. The first explored the regulation of CRABP mRNA levels in selected rat tissues by dietary retinoid status, and the relationship between CRABP mRNA and protein levels in different tissues. The second examined the cellular localization of CRABP expression in the testis. In order to conduct these experiments, a cDNA encoding CRABP was isolated and characterized. The DNA sequence of the coding region had 96% identity with that of the mouse CRABP cDNA and encodes a protein identical to mouse and bovine CRABP. CRABP mRNA and protein levels were quantified in five tissues from normal, retinoid-deficient, and retinol-repleted rats. Tissue CRABP and CRABP mRNA levels were highly correlated (P less than 0.01) indicating that inter-tissue variability of CRABP levels mainly results from regulation of CRABP mRNA levels. Neither CRABP protein nor mRNA levels were affected by retinol deficiency, in marked contrast with results previously demonstrated with cellular retinol-binding protein (CRBP) (J. Lipid Res. 1990. 31: 821-829). 35S-labeled CRABP cRNA probes were used to localize CRABP mRNA within the testis of adult rats by in situ hybridization. CRABP mRNA was localized selectively in the periphery of the seminiferous tubules, primarily in type A spermatogonia. The localization of CRABP mRNA differs from that of CRABP protein, which is known to be enriched in maturing and more mature germinal cells. This difference suggests that CRABP in germ cells may be highly stable, remaining in the maturing germ cells without degradation long after CRABP mRNA levels have declined to very low levels. The specific localization of CRABP mRNA and protein presumably reflects the biological roles of retinoic acid in the development and/or later function of germinal cells.     

Breast cancer cells express cathepsins B and L but not cathepsins K or H

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.     

Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney

To examine the correlation of localization of prorenin, renin, and cathepsins B, H, and L, immunocytochemistry was applied to rat renal tissue, using a sequence-specific anti-body (anti-prorenin) that recognizes the COOH terminus of the rat renin prosegment. In serial semi-thin sections, immunodeposits for prorenin, renin, and cathepsins B, H, and L were localized in the same juxtaglomerular (JG) cells. Immunodeposits for renin were detected throughout the cytoplasm of the cells, whereas those for prorenin were detected in the perinuclear region. Immunoreactivity for cathepsin B was stronger than that for cathepsins H and L. By electron microscopy, prorenin was localized in small (immature) granules but not in large mature granules, whereas renin was localized mainly in mature granules. In serial thin sections, prorenin, renin, and cathepsin B were colocalized in the same immature granules containing heterogeneously dense material (intermediate granules). By double immunostaining, co-localization of renin with cathepsins B, H, or L was demonstrated in mature granules. The results suggest the possibility that processing of prorenin to renin occurs in immature granules of rat JG cells, and cathepsin B detected in JG cells may be a major candidate for the maturation of renin.     

Expression and localization of cathepsin k in adult rat sertoli cells

The cathepsins are a family of cysteine proteases that have been broadly implicated in proteolytic processes during cell growth, cell development, and normal adult cellular function. Cathepsin L is a major secretory product of rat and mouse Sertoli cells, the absence of which in furless mice is associated with atrophy of some seminiferous tubules. However, furless mice produce viable sperm, suggesting the possibility that other members of the cathepsin family of proteases may complement cathepsin L action in the testis. Our objective herein was to begin to test this hypothesis. To this end, we first utilized cDNA microarray technology to identify the members of the cathepsin gene family expressed by freshly isolated adult rat Sertoli cells. This approach, complemented by Northern blot analyses, showed that in addition to cathepsin L, cathepsin K is highly and specifically expressed in Sertoli cells. As is also true of cathepsin L, cathepsin K mRNA was found to be expressed by Sertoli cells at specific stages of the cycle of the seminiferous epithelium, with maximal expression at stages VI-VII. The use of immunocytochemical methods revealed that cathepsin K protein localizes to the cytoplasm of Sertoli cells at stages VI-VIII, to small punctuate lysosomes at stages I-VIII and XIII-XIV, and to early and late residual bodies at stages IX-XII. This localization was found to be similar to that of cathepsin L. The similarity in the expression and localization of cathepsin K and cathepsin L suggest that the two proteases may have similar functions. If true, this might explain the fertility of furless mice. Further, the results suggest that cathepsin K, in both its secreted and lysosomal forms, may play a role in the degradation of Sertoli cell residual bodies.     

Cysteine proteinases in chondrosarcomas

The aim of the present study was to define the role of cathepsins B, H, K, L and S in the pathogenesis of human chondrosarcomas. For this purpose 40 tumour samples obtained from 12 patients with the diagnosis of conventional chondrosarcoma were systematically investigated for the expression of cathepsin mRNAs by Northern hybridisation, and for immunohistochemical localisation of the proteins. Northern analysis demonstrated the highest levels of cathepsins B and L in a recurring grade 1 chondrosarcoma, and in a grade 3 chondrosarcoma and in fibrous histiocytomas. Increased expression of cathepsin K mRNA was seen in seven chondrosarcomas, as well as in control tumours; fibrous histiocytomas, osteosarcomas, enchondromas and a giant cell tumour of bone. Cathepsin L was immunolocalised within the large chondrocytes, while cathepsin K was predominantly localised in large multinucleated osteoclastic cells and in some hypertrophic chondrocytes. These results suggest that chondrosarcoma can be included in the growing list of tumours, where cathepsins may well be involved in tumour progression. The simultaneous upregulation of cathepsins B and L, together with matrix metalloproteinase-13, and the association of cathepsin K with negative prognostic parameters suggests that an aggressive biological behaviour of chondrosarcoma may be related to the synthesis of cysteine proteinases and activation of other proteolytic enzymes. If this turns out to be the case, cathepsin inhibitors could provide the much needed adjuvant therapy for chondrosarcomas.     

Different immunolocalizations of cathepsins B, H, and L in the liver

Different localizations of cathepsin B, H, and L in normal rat liver were revealed immunohistochemically with anticathepsin Fab'-horseradish peroxidase conjugates. Staining of cathepsin B was strong in the periportal sinusoids, possibly in Kupffer cells; and weaker in panlobular hepatocytes. Staining of cathepsin H was strong in panlobular hepatocytes, especially in the periphery of the cytoplasm, possibly representing the peribiliary dense bodies; and weaker in periportal sinusoidal cells, possibly Kupffer cells. Staining of cathepsin L was strongest in centrilobular hepatocytes and weaker in periportal sinusoidal cells, possibly Kupffer cells. These findings, revealed for the first time in the present study, show that the histologic and intracellular localizations of the three cathepsins are different, suggesting that they have different roles in degradation of exogenous and endogenous proteins.     

家蚕组织蛋白酶基因家族的鉴定及表达特征分析

家蚕是鳞翅目完全变态昆虫,在其变态过程中伴随着巨大的形态变化,包括旧组织的解离和新组织的形成,在这过程中有多种组织蛋白酶参与。组织蛋白酶是一类细胞内蛋白酶,广泛存在于各个物种中,包括组织蛋白酶B、H、L等几个亚家族。对家蚕组织蛋白酶的研究将有利于阐明家蚕变态发育的详细过程。通过对家蚕基因组数据库进行筛选,共在家蚕中鉴定到13种组织蛋白酶,并对这13种组织蛋白酶的基本信息和表达模式进行了分析。另外,利用家蚕基因芯片数据和荧光定量PCR分析,鉴定编号为BGIBMGA004622的基因为卵巢特异表达的组织蛋白酶L亚家族基因。该基因全长1 209 bp,编码402个氨基酸。经过序列分析,该酶与其他物种的组织蛋白酶L具有较高的同源性,其活性位点高度保守,且与鳞翅目的组织蛋白酶L在进化上聚为一支。同时,对该基因进行克隆并原核表达,结果显示重组蛋白以包涵体的形式表达。定量PCR结果显示,该酶在蛹发育初期表达量逐渐升高,至蛹3 d达到最高值,推测其可能参与卵巢与卵母细胞的发育过程。     

Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation

The cathepsins B, H and L, lysosomal cysteine proteinases, play a major role in intracellular protein degradation. These proteinase activities and expressions were examined in a Ca2+ regulated epidermal culture system which consists of two morphological cell types: undifferentiated cells grown in low Ca2+ (0.1 mM concentration) and differentiated cells grown in high Ca2+ (1.8 mM concentration), respectively. Cathepsin B and L activities of the differentiated cells showed a several-fold increase compared to that of the undifferentiated cells. In addition, by using CM-cellulose column chromatography, cathepsin B and L were separated and the level of cathepsin L activity increased significantly. Cathepsin B, L and H were also detected by using an immunoblotting procedure in which their bands were expressed after differentiation was induced by the increasing calcium concentration. Cathepsin L activity and immunostaining intensity reached a maximum at 1 or 2 days of differentiation. In contrast, cystatin alpha (an endogenous inhibitor of cysteine-dependent cathepsins) appeared in the final stage of differentiation. These results indicate that the expression of epidermal cathepsins and their endogenous inhibitor are involved in part of the program of cell differentiation and the terminal differentiation process in cultured rat keratinocytes.     

Roles of cathepsins in reperfusion-induced apoptosis in cultured astrocytes

Astrocytic apoptosis may play a role in the central nervous system injury. We previously showed that reperfusion of cultured astrocytes with normal medium after exposure to hydrogen peroxide (H(2)O(2))-containing medium causes apoptosis. This study examines the involvement of the lysosomal enzymes cathepsins B and D in the astrocytic apoptosis. Reperfusion after exposure to H(2)O(2) caused a marked increase in caspase-3 and cathepsin D activities and a marked decrease in cathepsin B activity. Pepstatin A, an inhibitor of cathepsin D, and acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-aldehyde (Ac-DMQD-CHO), a specific inhibitor of caspase-3, blocked the H(2)O(2)-induced decrease in cell viability and DNA ladder formation in cultured rat astrocytes. The (L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA074 Me), a specific inhibitor of cathepsin B, did not affect the H(2)O(2)-induced cell injury. On the other hand, CA074 Me decreased cell viability with DNA ladder formation when cultured in the presence of Ac-DMQD-CHO. This caspase-independent apoptosis was attenuated by the addition of the cathepsin D inhibitor pepstatin A. Caspase-3 like activity was markedly inhibited by Ac-DMQD-CHO and partially by pepstatin A. Pepstatin A and CA074 Me inhibited cathepsin B and cathepsin D activities, respectively, in the presence and absence of Ac-DMQD-CHO. These results suggest that cathepsins B and D are involved in astrocytic apoptosis: cathepsin D acts as a death-inducing factor upstream of caspase-3 and the caspase-independent apoptosis is regulated antagonistically by cathepsins B and D.     

Quantitation and immunohistochemical localization of cathepsins E and D in rat tissues and blood cells

The distribution of cathepsins E and D in various rat tissues and blood cells was determined by immunoprecipitation and by immunohistochemistry with discriminative antibodies specific for each enzyme. While cathepsin D was detected in all of the tissues and blood cells tested (except for erythrocytes), cathepsin E had a relatively limited distribution. The cathepsin E content was highest in the stomach and was succeeded in the following order by the urinary bladder, thymus, spleen, cervical lymph node and bone marrow. Significant amounts of cathepsin E were also found in the colon, rectum, jejunum, skin, lung, kidney and submandibular gland. The other tissues tested had little or no detectable cathepsin E content. Of the blood cells tested, lymphocytes and peritoneal neutrophils contained high levels of cathepsin E. Erythrocytes had cathepsin E only as aspartic proteinases. When the subcellular localization of cathepsin E in the neutrophils was investigated by fractionation of the postnuclear supernatants, the enzyme behaved as a soluble cytosolic enzyme. In contrast, cathepsin D was mainly associated with the granular fraction. The immunohistochemical localization of cathepsins E and D was clearly different in the stomach, large intestines, kidney and urinary bladder, but was similar in the lymph node and spleen. The tissue-fixed macrophages, which were notable in the skeletal and cardiac muscle tissues, submucosal layers of the gastrointestinal tracts, salivary gland, lung and trachea, also exhibited similar intense immunoreactivities demonstrative of both cathepsins E and D.