Abstract
Hookworms are blood-feeding intestinal parasites of mammalian hosts and are one of the major human ailments affecting approximately 600 million people worldwide. These parasites form an intimate association with the host and are able to avoid vigorous immune responses in many ways including skewing of the response phenotype to promote parasite survival and longevity. The primary interface between the parasite and the host is the excretory/secretory component, a complex mixture of proteins, carbohydrates, and lipids secreted from the surface or oral openings of the parasite. The composition of this complex mixture is for the most part unknown but is likely to contain proteins important for the parasitic lifestyle and hence suitable as drug or vaccine targets. Using a strategy combining the traditional technology of one-dimensional SDS-PAGE and the newer fractionation technology of OFFGEL electrophoresis we identified 105 proteins from the excretory/secretory products of the blood-feeding stage of the dog hookworm, Ancylostoma caninum. Highly represented among the identified proteins were lectins, including three C-type lectins and three beta-galactoside-specific S-type galectins, as well as a number of proteases belonging to the three major classes found in nematodes, aspartic, cysteine, and metalloproteases. Interestingly 28% of the identified proteins were homologous to activation-associated secreted proteins, a family of cysteine-rich secreted proteins belonging to the sterol carrier protein/Tpx-1/Ag5/PR-1/Sc-7 (TAPS) superfamily. Thirty-four of these proteins were identified suggesting an important role in host-parasite interactions. Other protein families identified included hyaluronidases, lysozyme-like proteins, and transthyretin-like proteins. This work identified a suite of proteins important for the parasitic lifestyle and provides new insight into the biology of hookworm infection.
Highlights
Hookworms are blood-feeding intestinal parasites of mammalian hosts and are one of the major human ailments affecting ϳ600 million people worldwide
ES products allow helminth parasites to escape a vigorous immunological response: this is achieved in numerous ways including skewing of the response phenotype to promote parasite survival and longevity [10]
In this study we provide a survey of ES proteins from the adult stage of the bloodfeeding canine hookworm, Ancylostoma caninum, using a traditional proteomics approach of 1D electrophoresis followed by in-gel digestion and MS analysis and directly compare and contrast the findings with those obtained when we utilized the novel technique of OGE
Summary
Electrophoresis and In-gel Digestion—Two 10-l aliquots of a 9 mg/ml solution of A. caninum ES products were incubated for 2 h at 37 °C with an equal volume of Laemmli sample buffer. The 20 mM DTT was removed, and the samples were alkylated by the addition of 1 M iodoacetamide to a final concentration of 50 mM and incubation at 22 °C in darkness for 40 min. ES products were either electrophoresed on an SDS-PAGE gel and digested in gel [1], fractionated in a protein OGE prior to alkylation and tryptic digest [2], or reduced and alkylated using DTT and iodoacetamide before tryptic digestion and peptide OGE [3]. The protein samples were digested by incubation with 1 g of trypsin for 5 h before lyophilization and mass spectral analysis. Putative glycosylation sites were predicted using the on-line version of Prosite [35]
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