Definition of the Complete Schistosoma mansoni Hemoglobinase mRNA Sequence and Gene Expression in Developing Parasites

Meanawy, M. A. El; Aji, Toshiki; Phillips, Nelson B.; Davis, Richard E.; Salata, Robert A.; Malhotra, Indu; McClain, Donald A.; Aikawa, Masamichi; Davis, Alan H.

doi:10.4269/ajtmh.1990.43.67

Cited by 35 publications

(17 citation statements)

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“…2), then the human enzyme has an 8-amino acid propeptide. The C-terminal processing of the legumains of plants and Schistosoma takes the form of removal of a segment of about 14 kDa (4,27). Cleavage of human legumain in the vicinity of residue 300 would give rise to a mature protein of about 31 kDa, consistent with our experimental data for the mass of the deglycosylated pig enzyme (Fig.…”

Section: Covalent Structures Of Mammalian Legumains-supporting

confidence: 80%

Cloning, Isolation, and Characterization of Mammalian Legumain, an Asparaginyl Endopeptidase

Chen

Dando

Rawlings

et al. 1997

Journal of Biological Chemistry

335

325

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Legumain is a cysteine endopeptidase that shows strict specificity for hydrolysis of asparaginyl bonds. The enzyme belongs to peptidase family C13, and is thus unrelated to the better known cysteine peptidases of the papain family, C1 (Rawlings, N. D., and Barrett, A. J. (1994) Methods Enzymol. 244, 461-486). To date, legumain has been described only from plants and a blood fluke, Schistosoma mansoni. We now show that legumain is present in mammals. We have cloned and sequenced human legumain and part of pig legumain. We have also purified legumain to homogeneity (2200-fold, 8% yield) from pig kidney. The mammalian sequences are clearly homologous with legumains from non-mammalian species. Pig legumain is a glycoprotein of about 34 kDa, decreasing to 31 kDa on deglycosylation. It is an asparaginyl endopeptidase, hydrolyzing Z-Ala-Ala-Asn-7-(4-methyl)coumarylamide and benzoyl-Asn-p-nitroanilide. Maximal activity is seen at pH 5.8 under normal assay conditions, and the enzyme is irreversibly denatured at pH 7 and above. Mammalian legumain is a cysteine endopeptidase, inhibited by iodoacetamide and maleimides, but unaffected by compound E64 (transepoxysuccinyl-L-leucylamido-(4-guanidino)butane). It is inhibited by ovocystatin (cystatin from chicken egg white) and human cystatin C with K i values < 5 nM. We discuss the significance of the discovery of a cysteine endopeptidase of a new family and distinctive specificity in man and other mammals.Cysteine peptidases form one of the major groups of proteolytic enzymes, and can be divided into about 30 separate families on the basis of their molecular structures (reviewed in Refs. 1 and 2). Three families of cysteine endopeptidases have been known to be represented in mammals. The most numerous are those of the papain family (C1), which include cathepsins B, H, L, S, and others. These are predominantly lysosomal enzymes, responsible for proteolysis in the lysosomal/endosomal system and also are secreted to act extracellularly. In the cytosolic fraction of the cell, there are members of the other two families of cysteine endopeptidases: the families of calpain (family C2) and caspase (previously interleukin 1␤-converting enzyme; C14). These peptidases mediate limited proteolysis of cytosolic substrates. We now report that the legumain family (C13) can be added to the list of mammalian cysteine endopeptidases.Legumain is the name that was given by Kembhavi et al. (3) to an endopeptidase that is present in many leguminous and other seeds, after they had isolated and characterized the enzyme from Vigna aconitifolia (moth bean). Legumain is specific for the hydrolysis of asparaginyl bonds. The amino acid sequence of legumain from Ricinus communis (castor bean) showed it to be homologous with an enzyme from the fluke Schistosoma mansoni (4). At that time, the fluke enzyme was of unknown specificity, but it has now been shown also to be an asparaginyl endopeptidase (5), active on the test substrate that had been introduced by Kembhavi et al.The appearance of sequences homolog...

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Section: Covalent Structures Of Mammalian Legumains-supporting

confidence: 80%

Cloning, Isolation, and Characterization of Mammalian Legumain, an Asparaginyl Endopeptidase

Chen

Dando

Rawlings

et al. 1997

Journal of Biological Chemistry

335

325

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“…The latter was already shown to be active in the gut [67]. This applies also to the selected hemoglobinase (Smp_075800), which was localized to the gut [68]. Venom allergen-like proteins (VALs) of platyhelminths are members of the SCP/TAPS (Sperm-Coating Protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily and hypothesized to play not only roles in spermatogenesis but also beyond [69], which led to the choice of VAL7 (Smp_070240).…”

Section: Resultsmentioning

confidence: 76%

Imatinib Treatment Causes Substantial Transcriptional Changes in Adult Schistosoma mansoni In Vitro Exhibiting Pleiotropic Effects

et al. 2014

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BackgroundSchistosome parasites cause schistosomiasis, one of the most important infectious diseases worldwide. For decades Praziquantel (PZQ) is the only drug widely used for controlling schistosomiasis. The absence of a vaccine and fear of PZQ resistance have motivated the search for alternatives. Studies on protein kinases (PKs) demonstrated their importance for diverse physiological processes in schistosomes. Among others two Abl tyrosine kinases, SmAbl1 and SmAbl2, were identified in Schistosoma mansoni and shown to be transcribed in the gonads and the gastrodermis. SmAbl1 activity was blocked by Imatinib, a known Abl-TK inhibitor used in human cancer therapy (Gleevec/Glivec). Imatinib exhibited dramatic effects on the morphology and physiology of adult schistosomes in vitro causing the death of the parasites.Methodology/Principal FindingsHere we show modeling data supporting the targeting of SmAbl1/2 by Imatinib. A biochemical assay confirmed that SmAbl2 activity is also inhibited by Imatinib. Microarray analyses and qRT-PCR experiments were done to unravel transcriptional processes influenced by Imatinib in adult schistosomes in vitro demonstrating a wide influence on worm physiology. Surface-, muscle-, gut and gonad-associated processes were affected as evidenced by the differential transcription of e.g. the gynecophoral canal protein gene GCP, paramyosin, titin, hemoglobinase, and cathepsins. Furthermore, transcript levels of VAL-7 and egg formation-associated genes such as tyrosinase 1, p14, and fs800-like were affected as well as those of signaling genes including a ribosomal protein S6 kinase and a glutamate receptor. Finally, a comparative in silico analysis of the obtained microarray data sets and previous data analyzing the effect of a TGFβR1 inhibitor on transcription provided first evidence for an association of TGFβ and Abl kinase signaling. Among others GCP and egg formation-associated genes were identified as common targets.Conclusions/SignificanceThe data affirm broad negative effects of Imatinib on worm physiology substantiating the role of PKs as interesting targets.

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“…We had considered it most likely that Sm31 is a h , , 1 . I Study of two Schistosoma mansoni proteins, Sm3l and Sm32 (Barrett et al, 1984), human lysosomal cathepsin B (Chan et al, 1986), S. mansoni Sm31 (Klinkert et al, 1989), chicken calpain (Ohno et al, 1984), Streptococcus pyogenes proteinase (Tai et al, 1976) and S. mansoni Sm32 (Klinkert et al, 1989;El Meanawy et al, 1990), Clostridium histolyticum clostripain (Gilles et al, 1983) and poliovirus 3C proteinase (Argos et al, 1984) are aligned so as to achieve maximal identity ( Figure 6). Cysteine-proteinase-related enzymes found in bacteria are represented by the proteinase of S. pyogenes and clostripain from C. histolyticum.…”

Section: Discussionmentioning

confidence: 99%

Expression and partial characterization of a cathepsin B-like enzyme (Sm31) and a proposed ‘haemoglobinase’ (Sm32) from Schistosoma mansoni

Götz¹,

Klinkert²

1993

Biochemical Journal

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Schistosoma mansoni protein Sm31 is a cysteine proteinase similar to mammalian lysosomal cathepsin B, proposed to be a key enzyme in schistosome metabolism. Protein Sm32 has been identified as a putative cysteine proteinase termed a ‘haemoglobinase’. Since neither Sm31 nor Sm32 have been completely purified, some controversy of the nature of the ‘true’ digestive enzyme still exists. By incubating a radiolabelled cysteine-proteinase active-site-directed synthetic inhibitor with total S. mansoni proteins, the target of inhibition was Sm31 and not Sm32. The selectivity and irreversibility of inactivation make affinity labelling an invaluable tool for exploring key differences among closely related enzymes and also for studying proteinase activity in a cellular environment. In order to confirm these results, we expressed the complete cDNA sequences of Sm31 and Sm32 in insect cells and analysed the recombinant gene products for proteolytic activities. Cell extracts containing S. mansoni cathepsin B, but not those expressing ‘haemoglobinase’, were demonstrated to cleave a synthetic substrate benzyloxycarbonyl-arginylarginylaminomethylcoumarin in fluorescence assays. Our findings confirm previous assertions that a cysteine proteinase resembling cathepsin B is the haemoglobinase involved in digestion of host proteins. Thus, the original proposal that Sm32 is a cysteine proteinase has not been verified, and its function remains unknown.

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Definition of the Complete Schistosoma mansoni Hemoglobinase mRNA Sequence and Gene Expression in Developing Parasites

Cited by 35 publications

References 0 publications

Cloning, Isolation, and Characterization of Mammalian Legumain, an Asparaginyl Endopeptidase

Cloning, Isolation, and Characterization of Mammalian Legumain, an Asparaginyl Endopeptidase

Imatinib Treatment Causes Substantial Transcriptional Changes in Adult Schistosoma mansoni In Vitro Exhibiting Pleiotropic Effects

Expression and partial characterization of a cathepsin B-like enzyme (Sm31) and a proposed ‘haemoglobinase’ (Sm32) from Schistosoma mansoni

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