Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.23.5 (cathepsin D)
4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This review covers the unique catalytic and molecular properties of three proteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteinase from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic amino acids at P1 and P'1 like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P1, which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp76 to Ser or Thr and deletion of Ser78, corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at 1. In addition, the double mutant T77D/G78(S)G79 of porcine pepsin was able to activate bovine trypsinogen to trypsin by the selective cleavage of the K6-I7 bond of trypsinogen. Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Deuterolysin is a member of a family of metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand. Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that contains both N- and O-linked sugar chains. Site-directed mutagenesis of the cpdS, cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. A. saitoi carboxypeptidase indicated that Ser153, Asp357, and His436 residues were essential for the enzymic catalysis. The N-glycanase released high-mannose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man10 GlcNAc2 and Man11GlcNAc2, were characterized. An acidic 1,2-alpha-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2-alpha-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutant capable of producing Man5GlcNAc2 human-compatible sugar chains on glycoproteins was constructed. An expression vector for 1,2-alpha-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production of human-compatible high mannose-type (Man5GlcNAc2) sugar chains in S. cerevisiae was described.
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PMID:Unique catalytic and molecular properties of hydrolases from Aspergillus used in Japanese bioindustries. 1083 Apr 77

Cathepsin B, a lysosomal cysteine protease, is synthesized as a glycoprotein with two N-linked oligosaccharide chains, one of which is in the propeptide region while the other is in the mature region. When cultured rat hepatocytes were labeled with [(32)P]phosphate, (32)P-labeled cathepsin B was immunoprecipitated only in the proform from cell lysates and medium. Either Endo H or alkaline phosphatase treatment of (32)P-labeled procathepsin B demonstrated the acquisition of a mannose 6-phosphate (Man 6-P) residue on high mannose type oligosaccharides. To identify the site of phosphorylation, immunoisolated (35)S- or (32)P-labeled procathepsin B was incubated with purified lysosomal cathepsin D, since cathepsin D cleaves 48 amino acid residues from the N-terminus of procathepsin B, in which one N-linked oligosaccharide chain was also included [Kawabata, T. et al. (1993) J. Biochem. 113, 389-394]. Treatment of intracellular (35)S-labeled procathepsin B with a molecular mass of 39-kDa with cathepsin D resulted in the production of the 31-kDa intermediate form, but the (32)P-label incorporated into procathepsin B disappeared after treatment with cathepsin D. These results indicate that the phosphorylation of procathepsin B is restricted to an oligosaccharide chain present in the propeptide region. Interestingly, cathepsin B sorting to lysosomes was not inhibited by NH(4)Cl treatment and about 90% of the intracellular procathepsin B initially phosphorylated was secreted into the medium without being dephosphorylated intracellularly, and did not bind significantly to cation-independent-Man 6-P receptor, suggesting the failure of Man 6-P-dependent transport of procathepsin B to lysosomes. Additionally, about 50% of the newly synthesized (35)S-labeled cathepsin B was retained in the cells in mature forms consisting of a 29-kDa single chain form and a 24-kDa two chain form, while part of the procathepsin B was associated with membranes in a Man 6-P-independent manner. Taken together, these results show that in rat hepatocytes, cathepsin B is targeted to lysosomes by an alternative mechanism(s) other than the Man 6-P-dependent pathway.
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PMID:Lysosomal cysteine protease, cathepsin B, is targeted to lysosomes by the mannose 6-phosphate-independent pathway in rat hepatocytes: site-specific phosphorylation in oligosaccharides of the proregion. 1087 56

Tenascin (TN) is an extracellular matrix glycoprotein expressed in areas of epithelial-mesenchymal interactions during embryogenesis and in neoplasia. We studied the expression of TN in a series of 35 squamous cell invasive carcinomas of the larynx, 13 in situ carcinomas, 41 cases of dysplasia, 10 papillomas and 18 cases of keratosis using the monoclonal antibody TN2 on paraffin-embedded tissue. TN expression was correlated with the expression of fibronectin, CD44 and cathepsin D (CD) proteins, with the proliferation indices Ki-67 and proliferating cell nuclear antigen (PCNA) as well as with conventional clinicopathological variables. Malignant tumours showed a significantly greater stromal TN staining than benign lesions. In invasive carcinomas, the immunoreactivity was statistically higher than that in situ (P=0.01), dysplastic lesions (P<0.0001), papillomas (P=0.004) and keratosis (P<0.0001). A statistically significant difference of TN expression between in situ and dysplastic lesions was observed (P=0.001). In invasive lesions, TN expression was statistically correlated with CD44 expression (P=0.02) and a trend for correlation with CD of tumour cells and fibronectin expression was found (P=0.06 and P=0.09, respectively). The relationship of TN expression with the histological grade and the proliferative activity was insignificant. In conclusion, stromal TN expression may be involved in the complex mechanism of development of laryngeal lesions and may help to predict the risk of progression of pre-cancerous lesions to cancer.
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PMID:Expression of the extracellular matrix protein tenascin in laryngeal epithelial lesions: correlation with fibronectin, CD44, cathepsin D and proliferation indices. 1091 72

CTLA-4 is expressed on the surface of activated T cells and negatively regulates T cell activation. Because a low-level expression of CTLA-4 on the cell surface is sufficient to induce negative signals in T cells, the surface expression of CTLA-4 is strictly regulated. We previously demonstrated that the association of CTLA-4 with the clathrin-associated adaptor complex AP-2 induces internalization of CTLA-4 and keeps the surface expression low. However, the mechanism to induce high expression on the cell surface upon stimulation has not yet been clarified. To address this, we investigated the intracellular dynamics of CTLA-4 by analyzing its localization and trafficking in wild-type and mutant CTLA-4-transfected Th1 clones. CTLA-4 is accumulated in intracellular granules, which we identified as lysosomes. CTLA-4 is degraded in lysosomes in a short period, and the degradation process may serve as one of the mechanisms to regulate CTLA-4 expression. Upon TCR stimulation, CTLA-4-containing lysosomes are secreted as proven by the secretion of cathepsin D and beta-hexosaminidase in parallel with the increase of surface expression of CTLA-4 and lysosomal glycoprotein 85, a lysosomal marker. These results suggest that the cell surface expression of CTLA-4 is up-regulated upon stimulation by utilizing a mechanism of secretory lysosomes in CD4(+)T cells.
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PMID:Regulation of cell surface expression of CTLA-4 by secretion of CTLA-4-containing lysosomes upon activation of CD4+ T cells. 1104 36

In a total of 77 patients with epithelial ovarian carcinomas, new biologic parameters [estrogen and progesterone receptor (ER, PR), DNA-ploidy (DP), S-phase fraction (Spf), cycling index (Ki67), Her2b/neu oncoprotein, epidermal growth factor receptor (EGF-R), cathepsin D and P170 glycoprotein] have been simultaneously detected and correlated to the clinical outcome [progression-free (PFI) and overall survival (OAS)] in a preliminary study. Apart from conventional prognosticators (age, stage, grade, residual tumor) and the postoperative serum marker Ca125, DP (P = 0.01), Spf (P = 0.009), Ki67 (P = 0.05) and PR (P = 0.01) could predict a short OAS (log rank test), whereas cathepsin D was of borderline significance only. Prognostic significance could be improved by using combinations of different factors [two markers of differentiation (DP, PR), one marker of proliferation (Spf) and one marker describing local tumor spread (cathepsin D)]. The difference in prognosis between patients with either all or three favorable tumor factors and patients with two to four unfavorable tumor factors reached a similar significance as can be obtained using FIGO stage as a prognostic factor (P = 0.007 for PFI, P = 0.0005 for OAS). These results were similar if early stages (FIGO I and II) were excluded. However, in a Cox regression analysis, including stage and residual tumor, this combination was significantly independent for PFI and OAS and could give additional information. Therefore, the large number of new biologic tumor markers could be restricted to only a few significant prognosticators to predict prognosis in primary epithelial ovarian carcinoma. In the future tumor characterizations may allow more individualized treatment with more aggressive, or even without, cytotoxic therapy after primary surgery.
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PMID:Combination of new biologic parameters as a prognostic index in epithelial ovarian carcinoma. 1157 53

By applying density gradient electrophoresis (DGE) to human macrophages infected with Mycobacterium bovis BCG, we were able to separate three different bacterial fractions representing arrested phagosomes, phagolysosomes and mycobacterial clumps. After further purification of the phagosomal population, we found that isolated phagosomes containing live BCG were arrested in maturation as they exhibited only low amounts of the lysosomal glycoprotein LAMP-1 and processing of the lysosomal hydrolase cathepsin D was blocked. In addition, low amounts of MHC class I and class II molecules and the absence of HLA-DM suggest sequestration of mycobacterial phagosomes from antigen-processing pathways. We further investigated the involvement of the actin-binding protein coronin in intracellular survival of mycobacteria and showed that human coronin, as well as F-actin, were associated with early stages of mycobacterial phagocytosis but not with phagosome maintenance. Therefore, we conclude that the unique DGE migration pattern of arrested phagosomes is not as a result of retention of coronin, but that there are other proteins or lipids responsible for the block in maturation in human macrophages.
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PMID:Coronin is involved in uptake of Mycobacterium bovis BCG in human macrophages but not in phagosome maintenance. 1173 91

Molecular mechanisms of endocytosis in the genetically and biochemically tractable professional phagocyte Dictyostelium discoideum reveal a striking degree of similarity to higher eukaryotic cells. Pulse-chase feeding with latex beads allowed purification of phagosomes at different stages of maturation. Gentle ATP stripping of an actin meshwork entrapping contaminating organelles resulted in a 10-fold increase in yield and purity, as confirmed by electron microscopy. Temporal profiling of signaling, cytoskeletal, and trafficking proteins resulted in a complex molecular fingerprint of phagosome biogenesis and maturation. First, nascent phagosomes were associated with coronin and rapidly received a lysosomal glycoprotein, LmpB. Second, at least two phases of delivery of lysosomal hydrolases (cathepsin D [CatD] and cysteine protease [CPp34]) were accompanied by removal of plasma membrane components (PM4C4 and biotinylated surface proteins). Third, a phase of late maturation, preparing for final exocytosis of undigested material, included quantitative recycling of hydrolases and association with vacuolin. Also, lysosomal glycoproteins of the Lmp family showed distinct trafficking kinetics. The delivery and recycling of CatD was directly visualized by confocal microscopy. This heavy membrane traffic of cargos was precisely accompanied by regulatory proteins such as the Rab7 GTPases and the endosomal SNAREs Vti1 and VAMP7. This initial molecular description of phagocytosis demonstrates the feasibility of a comprehensive analysis of phagosomal lipids and proteins in genetically modified strains.
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PMID:High-resolution dissection of phagosome maturation reveals distinct membrane trafficking phases. 1238 53

alpha 2-Macroglobulin (alpha 2M) is an abundant glycoprotein with the intrinsic capacity for capturing diverse proteins for rapid delivery into cells. After internalization by the receptor- mediated endocytosis, alpha 2M-protein complexes were rapidly degraded in the endolysosome system. Although this is an important pathway for clearance of both alpha 2M and biological targets, little is known about the nature of alpha 2M degradation in the endolysosome system. To investigate the possible involvement of intracellular aspartic proteinases in the disruption of structural and functional integrity of alpha 2M in the endolysosome system, we examined the capacity of alpha 2M for interacting with cathepsin E and cathepsin D under acidic conditions and the nature of its degradation. alpha 2M was efficiently associated with cathepsin E under acidic conditions to form noncovalent complexes and rapidly degraded through the generation of three major proteins with apparent molecular masses of 90, 85 and 30 kDa. Parallel with this reaction, alpha 2M resulted in the rapid loss of its antiproteolytic activity. Analysis of the N-terminal amino-acid sequences of these proteins revealed that alpha 2M was selectively cleaved at the Phe811-Leu812 bond in about 100mer downstream of the bait region. In contrast, little change was observed for alpha 2M treated by cathepsin D under the same conditions. Together, the synthetic SPAFLA peptide corresponding to the Ser808-Ala813 sequence of human alpha 2M, which contains the cathepsin E-cleavage site, was selectively cleaved by cathepsin E, but not cathepsin D. These results suggest the possible involvement of cathepsin E in disruption of the structural and functional integrity of alpha 2M in the endolysosome system.
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PMID:Disruption of structural and functional integrity of alpha 2-macroglobulin by cathepsin E. 1263 Dec 77

Endocytosis, whereby proteins are internalized from the epididymal lumen to be eventually degraded in lysosomes, is one of the major functions of the epididymal epithelial cells in maintaining a proper luminal milieu conducive for sperm maturation. In the present study, using light microscope immunocytochemical methods, we examined the regulation of 2 lysosomal enzymes, sulfated glycoprotein-1 (SGP-1) and cathepsin D, in adult rat epididymides fixed in Bouin fixative and embedded in paraffin. After orchidectomy (O) with or without testosterone (T) supplementation, efferent duct ligation (EDL), or hypophysectomy (H), lysosomes of principal cells were intensely reactive with the anti-SGP-1 antibody, as were narrow, clear, and basal cells, with staining patterns similar to that of control animals. These experimental procedures also had no effect on cathepsin D expression in all cell types, except for clear cells of the corpus and cauda epididymidis, which after orchiedectomy and hypophysectomy, became intensely reactive, unlike their completely unreactive state in control animals. In O+T animals, as well as in EDL animals, clear cells remained unreactive. These data taken together suggest that expression of SGP-1 is not under the control of testicular or pituitary factors, as is also the case for cathepsin D expression by principal, narrow, and basal cells. However, specific inhibition of cathepsin D expression by testosterone or one of its metabolites appears to occur in clear cells of the corpus and cauda epididymidis. Furthermore, in addition to small, typical lysosomes, principal cells also revealed large supranuclear and infranuclear spherical structures that were immunoreactive with both anti-SGP-1 and anti-cathepsin D antibodies, suggesting their lysosomal nature. With electron microscopy, these structures appeared electron-lucent and contained membranous profiles embedded in an electron-dense, granular background. Such images suggest that the various experimental procedures adversely affect the expression of several other lysosomal enzymes in principal cells, leading to a lysosomal phenotype similar to that observed in various lysosomal storage diseases.
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PMID:Regulation of sulfated glycoprotein-1 and cathepsin D expression in adult rat epididymis. 1272 Dec 18

Fibulin-1 (Fbln-1) is an immunogenic breast cancer-related glycoprotein identified by serological analysis of cDNA expression library (SEREX) strategy. Here, we show that dendritic cells from two breast cancer patients elicited a CD4(+)-mediated T-cell response to Fbln-1 presentation. In both patients, an antibody response to Fbln-1 was also found. By contrast, a Fbln-1-seronegative patient and a weakly seropositive patient demonstrated no such T-cell response. Analysis of human breast cancers for Fbln-1 RNA and protein expression revealed the presence of Fbln-1C and -1D variants. Fbln-1 was detected in the cytoplasm and at the cell surface of different human breast carcinoma cell lines. Immunohistochemical analysis of 528 archival primary breast carcinomas showed the expression of Fbln-1 in 35% of the cases. When the immunohistochemical findings were compared against pathobiological information associated with each specimen, an inverse relationship between Fbln-1 and cathepsin D expression was observed (P=0.04). Furthermore, even though long-term survival was similar between Fbln-1-positive and -negative cases, the survival of Fbln-1-positive cases improved when a lymphoid infiltrate was present at the tumour site. Taken together, our findings of an Fbln-1-specific immunity and the improved survival associated with Fbln-1 expression in the presence of lymphoid infiltration point to a role of Fbln-1 in tumour immunosurveillance.
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PMID:Immunological and pathobiological roles of fibulin-1 in breast cancer. 1469 54


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