Acasp,a Gene Encoding a Cathepsin D-like Aspartic Protease from the HookwormAncylostoma caninum

Harrop, Stephen Anthony; Prociv, Paul; Brindley, Paul J.

doi:10.1006/bbrc.1996.1503

Cited by 46 publications

(32 citation statements)

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“…Clawed lobster cathepsin D1 has the pro-enzyme sequence (Fig. 3), but lacks a sequence indicating the presence of the β-hairpin loop; this seems to be a common feature of invertebrate cathepsins D (Boldbaatar et al 2006;Cho and Raikhel 1992;Harrop et al 1996) and suggests an alternative activation mechanism, yet to be investigated.…”

Section: Zymogen-activationmentioning

confidence: 99%

Aspartic Cathepsin D Endopeptidase Contributes to Extracellular Digestion in Clawed Lobsters Homarus americanus and Homarus gammarus

et al. 2010

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Acid digestive proteinases were studied in the gastric fluids of two species of clawed lobster (Homarus americanus and Homarus gammarus). An active protein was identified in both species as aspartic proteinase by specific inhibition with pepstatin A. It was confirmed as cathepsin D by mass mapping, N-terminal, and full-length cDNA sequencing. Both lobster species transcribed two cathepsin D mRNAs: cathepsin D1 and cathepsin D2. Cathepsin D1 mRNA was detected only in the midgut gland, suggesting its function as a digestive enzyme. Cathepsin D2 mRNA was found in the midgut gland, gonads, and muscle. The deduced amino acid sequence of cathepsin D1 and cathepsin D2 possesses two catalytic DTG active-site motifs, the hallmark of aspartic proteinases. The putatively active cathepsin D1 has a molecular mass of 36.4 kDa and a calculated pI of 4.14 and possesses three potential glycosylation sites. The sequences showed highest similarities with cathepsin D from insects but also with another crustacean cathepsin D. Cathepsin D1 transcripts were quantified during a starvation period using real-time qPCR. In H. americanus, 15 days of starvation did not cause significant changes, but subsequent feeding caused a 2.5-fold increase. In H. gammarus, starvation caused a 40% reduction in cathepsin D1 mRNA, and no effect was observed with subsequent feeding.

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Section: Zymogen-activationmentioning

confidence: 99%

Aspartic Cathepsin D Endopeptidase Contributes to Extracellular Digestion in Clawed Lobsters Homarus americanus and Homarus gammarus

et al. 2010

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“…Moreover, the intestinal contents from the related blood-feeding helminth, Schistosoma mansoni, are acidic pH (11). mRNAs encoding hookworm proteases of different mechanistic classes, including aspartic (19,20,23), cysteine (24), and metalloproteases (25,26), have been cloned, and the expression sites of some have been localized to the intestinal brush border of the blood-feeding adult stage (for a review, see Ref. 18).…”

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confidence: 99%

A Multi-enzyme Cascade of Hemoglobin Proteolysis in the Intestine of Blood-feeding Hookworms

Williamson¹,

Lecchi

Turk

et al. 2004

Journal of Biological Chemistry

157

115

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Blood-feeding pathogens digest hemoglobin (Hb) as a source of nutrition, but little is known about this process in multicellular parasites. The intestinal brush border membrane of the canine hookworm, Ancylostoma caninum, contains aspartic proteases (APR-1), cysteine proteases (CP-2), and metalloproteases (MEP-1), the first of which is known to digest Hb. We now show that Hb is degraded by a multi-enzyme, synergistic cascade of proteolysis. Recombinant APR-1 and CP-2, but not MEP-1, digested native Hb and denatured globin. MEP-1, however, did cleave globin fragments that had undergone prior digestion by APR-1 and CP-2. Proteolytic cleavage sites within the Hb ␣ and ␤ chains were determined for the three enzymes, identifying a total of 131 cleavage sites. By scanning synthetic combinatorial peptide libraries with each enzyme, we compared the preferred residues cleaved in the libraries with the known cleavage sites within Hb. The semi-ordered pathway of Hb digestion described here is surprisingly similar to that used by Plasmodium to digest Hb and provides a potential mechanism by which these hemoglobinases are efficacious vaccines in animal models of hookworm infection.

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“…However, the breakdown of these macromolecules by an aspartyl proteinase is unusual and has not been described previously, although an aspartyl proteinase activity has been demonstrated in the secretions of adult Haemonchus contortus, 24 and a cDNA encoding a cathepsin D-like aspartyl proteinase has recently been identified from the dog hookworm Ancylostoma caninum. 25 These data suggest that N. americanus larvae primarily facilitate skin penetration by the use of a mixture aspartyl and metalloproteases, with a serine protease contributing to the breakdown of elastin. Similarly, a potent histolytic metalloprotease capable of degrading elastin and fibronectin but not type I collagen has been described in the secretions of Strongyloides stercoralis.…”

Section: Discussionmentioning

confidence: 85%

Necator americanus (human hookworm) aspartyl proteinases and digestion of skin macromolecules during skin penetration.

Brown¹,

Girod²,

Billett³

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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Abstract. The infective larvae of Necator americanus were shown to secrete all mechanistic classes of proteolytic enzymes with two overall pH optima of 6.5 and 8.5 using fluorescein isothiocyanate-labeled casein as the substrate. Since infective larvae are obligate skin penetrators, the effect of each of these enzyme classes against macromolecules derived from human skin was examined. Larval secretions were shown to degrade collagen types I, III, IV, and V, fibronectin, laminin, and elastin. All the skin macromolecules tested were hydrolyzed by aspartyl proteinase activity, which was inhibitable by pepstatin A. Collagen and elastin was also hydrolyzed by metalloproteinase activity, while the serine proteinase activity hydrolyzed only elastin. As a consequence of these experiments, the effect of proteinase inhibitors on the penetration of live larvae through hamster skin was tested. Larval penetration was significantly inhibited only by pepstatin A, confirming the importance of the aspartyl proteinase activity during the skin penetration process.Necator americanus is a human pathogen that invades the body by penetrating the skin. In 1982 Matthews 1 showed that cellular destruction of the skin during larval penetration through the epidermis was mediated by an undefined enzymatic process, optimally active at pH 8. Subsequently, Salafsky and others 2 developed a gelatin-agar membrane as a model for hookworm skin penetration and showed that serine, and possibly cysteinyl, proteinase activities were responsible for larval penetration through this type of membrane. The presence of cysteinyl and serine proteinases in larval secretions was confirmed using gelatin substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 3 and it has been further demonstrated that live N. americanus larvae secrete a gelatinolytic metalloproteinase proteinase. 4 Despite the obvious presence of proteolytic enzymes in the excretory-secretory (ES) products of N. americanus larvae, an exact role for each of these proteinases has yet to be defined. Consequently, and given the fact that the larvae are obligate skin penetrators, we now describe attempts to assign a role during skin penetration for the previously described proteinases secreted by N. americanus larvae. The hydrolysis of a number of skin macromolecules has been studied and the enzymes responsible for their degradation have been implicated using standard proteinase inhibitors. Despite being a human parasite, N. americanus can also be maintained in the golden hamster. 5 Therefore, to confirm the data obtained using individual skin macromolecules, the effects of proteinase inhibitors on the penetration of live larvae through excised hamster skin was also studied. MATERIALS AND METHODSPreparation of N. americanus larval ES products. Necator americanus was maintained in syngeneic DSN hamsters as described by Sen and Seth. 5 Infective larvae were cultured from fecal material by a method modified from Harada and Mori 6 and previously described by Kumar and Pritchard. ...

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Acasp,a Gene Encoding a Cathepsin D-like Aspartic Protease from the HookwormAncylostoma caninum

Cited by 46 publications

References 0 publications

Aspartic Cathepsin D Endopeptidase Contributes to Extracellular Digestion in Clawed Lobsters Homarus americanus and Homarus gammarus

Aspartic Cathepsin D Endopeptidase Contributes to Extracellular Digestion in Clawed Lobsters Homarus americanus and Homarus gammarus

A Multi-enzyme Cascade of Hemoglobin Proteolysis in the Intestine of Blood-feeding Hookworms

Necator americanus (human hookworm) aspartyl proteinases and digestion of skin macromolecules during skin penetration.

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