Identification of Secreted Cysteine Proteases from the Parasitic Nematode Haemonchus contortus Detected by Biotinylated Inhibitors

Division of Infection Biology, Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan, 1, 3584CL, Utrecht, The Netherlands.
Infection and Immunity (Impact Factor: 3.73). 04/2006; 74(3):1989-93. DOI: 10.1128/IAI.74.3.1989-1993.2006
Source: PubMed


Seven cathepsin B-like cysteine proteases (CBLs) were identified from the immunoprotective excretory-secretory products of
Haemonchus contortus. Two-dimensional (2-D) zymography and biotinylated inhibitors were employed to localize active CBLs in 2-D protein gels.
Mass spectrometry provided the identification of AC-4, HMCP1, HMCP2, and GCP7 as well as three novel CBLs encoded by clustered
expressed sequence tags.

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Available from: Albert J R Heck, Feb 01, 2014
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    • "These include both novel and previously identified cbl genes (AC-4, gcp-7 and hmcp-2) [72-74], while other cbl transcripts are significantly up-regulated in the L4 stage and in adult male worms, but not in the gut, suggesting a role in development and reproduction rather than feeding. The CBLs identified here contain a putative amino-terminal signal peptide and several have been identified in adult worm excretory-secretory products [75]. It has also been proposed that sequence variation of Hc-CBLs may confer antigenic diversity [76], and presentation to the host immune system through secretion may therefore drive the maintenance of the diversity of Hc-cbl genes. "
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    ABSTRACT: The small ruminant parasite Haemonchus contortus is the most widely used parasitic nematode in drug discovery, vaccine development and anthelmintic resistance research. Its remarkable propensity to develop resistance threatens the viability of the sheep industry in many regions of the world and provides a cautionary example of the effect of mass drug administration to control parasitic nematodes. Its phylogenetic position makes it particularly well placed for comparison with the free-living nematode Caenorhabditis elegans and the most economically important parasites of livestock and humans. Here we report the detailed analysis of a draft genome assembly and extensive transcriptomic dataset for H. contortus. This represents the first genome to be published for a strongylid nematode and the most extensive transcriptomic dataset for any parasitic nematode reported to date. We show a general pattern of conservation of genome structure and gene content between H. contortus and C. elegans, but also a dramatic expansion of important parasite gene families. We identify genes involved in parasite-specific pathways such as blood feeding, neurological function, and drug metabolism. In particular, we describe complete gene repertoires for known drug target families, providing the most comprehensive understanding yet of the action of several important anthelmintics. Also, we identify a set of genes enriched in the parasitic stages of the lifecycle and the parasite gut that provide a rich source of vaccine and drug target candidates. The H. contortus genome and transcriptome provides an essential platform for postgenomic research in this and other important strongylid parasites.
    Genome biology 08/2013; 14(8):R88. DOI:10.1186/gb-2013-14-8-r88 · 10.81 Impact Factor
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    • "Tissue migration and other interactions with host cells may be facilitated by the function of these enzymes, by mediating or changing proteolytic functions [53]. Several studies have considered these enzymes as important therapeutic targets for parasite control [54-56,93]. Results from the pathway analysis carried out using KOBAS were compared with the results obtained using KAAS. "
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    ABSTRACT: Background Teladorsagia circumcincta (order Strongylida) is an economically important parasitic nematode of small ruminants (including sheep and goats) in temperate climatic regions of the world. Improved insights into the molecular biology of this parasite could underpin alternative methods required to control this and related parasites, in order to circumvent major problems associated with anthelmintic resistance. The aims of the present study were to define the transcriptome of the adult stage of T. circumcincta and to infer the main pathways linked to molecules known to be expressed in this nematode. Since sheep develop acquired immunity against T. circumcincta, there is some potential for the development of a vaccine against this parasite. Hence, we infer excretory/secretory molecules for T. circumcincta as possible immunogens and vaccine candidates. Results A total of 407,357 ESTs were assembled yielding 39,852 putative gene sequences. Conceptual translation predicted 24,013 proteins, which were then subjected to detailed annotation which included pathway mapping of predicted proteins (including 112 excreted/secreted [ES] and 226 transmembrane peptides), domain analysis and GO annotation was carried out using InterProScan along with BLAST2GO. Further analysis was carried out for secretory signal peptides using SignalP and non-classical sec pathway using SecretomeP tools. For ES proteins, key pathways, including Fc epsilon RI, T cell receptor, and chemokine signalling as well as leukocyte transendothelial migration were inferred to be linked to immune responses, along with other pathways related to neurodegenerative diseases and infectious diseases, which warrant detailed future studies. KAAS could identify new and updated pathways like phagosome and protein processing in endoplasmic reticulum. Domain analysis for the assembled dataset revealed families of serine, cysteine and proteinase inhibitors which might represent targets for parasite intervention. InterProScan could identify GO terms pertaining to the extracellular region. Some of the important domain families identified included the SCP-like extracellular proteins which belong to the pathogenesis-related proteins (PRPs) superfamily along with C-type lectin, saposin-like proteins. The 'extracellular region' that corresponds to allergen V5/Tpx-1 related, considered important in parasite-host interactions, was also identified. Six cysteine motif (SXC1) proteins, transthyretin proteins, C-type lectins, activation-associated secreted proteins (ASPs), which could represent potential candidates for developing novel anthelmintics or vaccines were few other important findings. Of these, SXC1, protein kinase domain-containing protein, trypsin family protein, trypsin-like protease family member (TRY-1), putative major allergen and putative lipid binding protein were identified which have not been reported in the published T. circumcincta proteomics analysis. Detailed analysis of 6,058 raw EST sequences from dbEST revealed 315 putatively secreted proteins. Amongst them, C-type single domain activation associated secreted protein ASP3 precursor, activation-associated secreted proteins (ASP-like protein), cathepsin B-like cysteine protease, cathepsin L cysteine protease, cysteine protease, TransThyretin-Related and Venom-Allergen-like proteins were the key findings. Conclusions We have annotated a large dataset ESTs of T. circumcincta and undertaken detailed comparative bioinformatics analyses. The results provide a comprehensive insight into the molecular biology of this parasite and disease manifestation which provides potential focal point for future research. We identified a number of pathways responsible for immune response. This type of large-scale computational scanning could be coupled with proteomic and metabolomic studies of this parasite leading to novel therapeutic intervention and disease control strategies. We have also successfully affirmed the use of bioinformatics tools, for the study of ESTs, which could now serve as a benchmark for the development of new computational EST analysis pipelines.
    BMC Genomics 12/2012; 13(7). DOI:10.1186/1471-2164-13-S7-S10 · 3.99 Impact Factor
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    • "Two rice cysteine protease inhibitors (OCI-OCII), with small differences in their target enzyme-binding site showed different degrees of affinity for Meloidogyne hapla cysteine proteases [1, 15]. Thus, variation in the pentapeptide sequence may determine differences in affinity for different proteases and may indicate that the previously identified [38] seven secreted cysteine proteases are absent from the H. contortus cystatin-1-bound fraction because of a low affinity for this inhibitor. Alternatively it cannot be excluded that many secreted CBL have a cystatin bound to their active site thus prohibiting their binding to the cystatin column. "
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    ABSTRACT: The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.
    Veterinary Research 05/2009; 40(4):41. DOI:10.1051/vetres/2009025 · 2.82 Impact Factor
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