Functional Expression of Dipeptidyl Peptidase I (Cathepsin C) of the Oriental Blood Fluke Schistosoma japonicum in Trichoplusia ni Insect Cells

Hola-Jamriska, L.; King, Lynette T.; Dalton, John P.; Mann, Victoria H.; Aaskov, John; Brindley, Paul J.

doi:10.1006/prep.2000.1261

Cited by 21 publications

(10 citation statements)

References 35 publications

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“…Flatworm CTSC expressed in insect cells also exhibited similar three bands of 55 kDa, 39 kDa, and 38 kDa polypeptides after purification. N-terminal sequencing demonstrated that the 39 kDa and 38 kDa polypeptides shared identical N-terminal sequence and were produced by removal of 126 residues from N-terminus of 55 kDa proenzyme [23]. Thus, it is likely that 31 kDa and 30 kDa polypeptides are derived from 51 kDa polypeptide by differential processing of N-terminus of peptides as demonstrated in flatworm rCTSC.…”

Section: Resultsmentioning

confidence: 99%

Expression and Purification of Active Recombinant Cathepsin C (Dipeptidyl Aminopeptidase I) of Kuruma Prawn Marsupenaeus japonicus in Insect Cells

Qiu

Feng

Yamano

2009

BioMed Research International

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Cathepsin C (CTSC) is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen) is localized exclusively in cortical rods (CRs) of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37°C for 40 hours under native conditions, the recombinant CTSC (rCTSC) exhibited increased activity against the synthetic substrate Gly-Phe-β-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.

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Section: Resultsmentioning

confidence: 99%

Expression and Purification of Active Recombinant Cathepsin C (Dipeptidyl Aminopeptidase I) of Kuruma Prawn Marsupenaeus japonicus in Insect Cells

Qiu

Feng

Yamano

2009

BioMed Research International

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“…These include the cysteine endopeptidases, cathepsin L, cathepsin B, asparaginyl endopeptidase (legumain), and a cathepsin D-like aspartyl endopeptidase [39]; the localization of these proteases in the gastrodermis has been confirmed by immunocytochemistry [38], [40]–[43]. Exopeptidases, such as dipeptidylpeptidase (cathepsin C) [44] and proline carboxypeptidase homologs have also been identified [11]. A leucine aminopeptidease (LAP), with optimal activity at neutral pH, has been immunolocalized to the gastrodermal cells lining the lumen of adult schistosomes; LAP-immunoreactivity was considerably stronger within the gut of females versus males [45].…”

Section: Feeding Via the Alimentary Tractmentioning

confidence: 97%

Schistosome Feeding and Regurgitation

et al. 2014

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Schistosomes are parasitic flatworms that infect >200 million people worldwide, causing the chronic, debilitating disease schistosomiasis. Unusual among parasitic helminths, the long-lived adult worms, continuously bathed in blood, take up nutrients directly across the body surface and also by ingestion of blood into the gut. Recent proteomic analyses of the body surface revealed the presence of hydrolytic enzymes, solute, and ion transporters, thus emphasising its metabolic credentials. Furthermore, definition of the molecular mechanisms for the uptake of selected metabolites (glucose, certain amino acids, and water) establishes it as a vital site of nutrient acquisition. Nevertheless, the amount of blood ingested into the gut per day is considerable: for males ∼100 nl; for the more actively feeding females ∼900 nl, >4 times body volume. Ingested erythrocytes are lysed as they pass through the specialized esophagus, while leucocytes become tethered and disabled there. Proteomics and transcriptomics have revealed, in addition to gut proteases, an amino acid transporter in gut tissue and other hydrolases, ion, and lipid transporters in the lumen, implicating the gut as the site for acquisition of essential lipids and inorganic ions. The surface is the principal entry route for glucose, whereas the gut dominates amino acid acquisition, especially in females. Heme, a potentially toxic hemoglobin degradation product, accumulates in the gut and, since schistosomes lack an anus, must be expelled by the poorly understood process of regurgitation. Here we place the new observations on the proteome of body surface and gut, and the entry of different nutrient classes into schistosomes, into the context of older studies on worm composition and metabolism. We suggest that the balance between surface and gut in nutrition is determined by the constraints of solute diffusion imposed by differences in male and female worm morphology. Our conclusions have major implications for worm survival under immunological or pharmacological pressure.

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“…A model for this catabolic process proposes that the initial cleavages of hemoglobin are performed by endopeptidases, including cathepsins B, L and D, liberating short peptides that are subsequently hydrolysed by exopeptidases into absorbable dipeptides and amino acids (Brindley et al, 1997;Tort et al, 1999;Sajid and McKerrow, 2002). Whereas we and others have characterised the exopeptidase dipeptidyl peptidase I, ( ¼ cathepsin C), which is secreted from the parasite gastrodermis into the gut and most likely is the enzyme that cleaves dipeptides from fragments of host hemoglobin (Hola-Jamriska et al, 1998, 2000, little is known of the putative aminopeptidase that is crucial to the final stage of liberating free amino acids.…”

Section: Introductionmentioning

confidence: 98%

Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum

McCarthy

Stack

Donnelly

et al. 2004

International Journal for Parasitology

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An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling. q

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Functional Expression of Dipeptidyl Peptidase I (Cathepsin C) of the Oriental Blood Fluke Schistosoma japonicum in Trichoplusia ni Insect Cells

Cited by 21 publications

References 35 publications

Expression and Purification of Active Recombinant Cathepsin C (Dipeptidyl Aminopeptidase I) of Kuruma Prawn Marsupenaeus japonicus in Insect Cells

Expression and Purification of Active Recombinant Cathepsin C (Dipeptidyl Aminopeptidase I) of Kuruma Prawn Marsupenaeus japonicus in Insect Cells

Schistosome Feeding and Regurgitation

Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum

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