The complexity of the secreted NPA and FAR lipid-binding protein families of Haemonchus contortus revealed by an iterative proteomics-bioinformatics approach.

Two different classes of small nematode specific lipid-binding proteins, the nematode polyprotein allergens/antigens (NPAs) and the fatty acid- and retinol-binding (FAR) proteins, are secreted by helminth parasites. Until now, there was no evidence of the expression or secretion of these two families of proteins in Haemonchus contortus. In this study, we applied proteomic and bioinformatic tools in an iterative manner to reveal the expression and complexity of these proteins in the excretory/secretory products (ESP) of adult H. contortus at the protein and gene levels. Initial examination of the mass spectra of ESP fractions against standard databases returned nine peptides mapping to Ostertagia ostertagi NPA and FAR sequences. Searches of the H. contortus EST and genomic contig databases with the O. ostertagi and Caenorhabditis elegans homologues retrieved diverse sequences encoding H. contortus NPA and FAR proteins. H. contortus sequences were then integrated into a customized database and a new search of the mass spectra achieved a 10-fold improvement in coverage of the predicted H. contortus NPAs. The final analyses of the mass spectra achieved 49-60% coverage of H. contortus NPAs and 7-47% coverage of H. contortus FARs. Moreover, the diversity in structures of the encoding genes was revealed by assembling the genomic sequence data with predicted protein sequences confirmed by the peptide evidence. We predict there are at least one Hc-NPA gene and six Hc-FAR genes in H. contortus, and life stage gene expression of Hc-FAR-1 to -6 revealed unique transcription patterns for each of these genes.

[1]  Lucinda K. Southworth,et al.  An elt-3/elt-5/elt-6 GATA Transcription Circuit Guides Aging in C. elegans , 2008, Cell.

[2]  R. Maizels,et al.  Brugia pahangi and Brugia malayi: a surface-associated glycoprotein (gp15/400) is composed of multiple tandemly repeated units and processed from a 400-kDa precursor. , 1993, Experimental parasitology.

[3]  Stephen E Von Stetina,et al.  The embryonic muscle transcriptome of Caenorhabditis elegans , 2007, Genome Biology.

[4]  R. Curi,et al.  Mechanisms by which fatty acids regulate leucocyte function. , 2007, Clinical science.

[5]  I. Hope,et al.  Caenorhabditis elegans reporter fusion genes generated by seamless modification of large genomic DNA clones , 2006, Nucleic acids research.

[6]  J. Barrett.  Biochemistry of parasitic helminths , 1982 .

[7]  N. C. Price,et al.  The FAR Protein Family of the Nematode Caenorhabditis elegans , 2003, The Journal of Biological Chemistry.

[8]  Sommerville Ri.  Influence of potassium ion and osomtic pressure on development of Haemonchus contortus in vitro. , 1976 .

[9]  J. Culpepper,et al.  Molecular characterization of a Dirofilaria immitis cDNA encoding a highly immunoreactive antigen. , 1992, Molecular and biochemical parasitology.

[10]  Jonathan E. Allen,et al.  Draft Genome of the Filarial Nematode Parasite Brugia malayi , 2007, Science.

[11]  K. Anbu,et al.  Identification of a 55 kDa Haemonchus contortus excretory/secretory glycoprotein as a neutrophil inhibitory factor , 2007, Parasite immunology.

[12]  J. Moore,et al.  Extensive diversity in repeat unit sequences of the cDNA encoding the polyprotein antigen/allergen from the bovine lungworm Dictyocaulus viviparus. , 1995, Molecular and biochemical parasitology.

[13]  E. Liebau,et al.  Conformational and functional analysis of the lipid binding protein Ag‐NPA‐1 from the parasitic nematode Ascaridia galli , 2004, The FEBS journal.

[14]  Andrew Smith.  Genome sequence of the nematode C-elegans: A platform for investigating biology , 1998 .

[15]  A. Larbi,et al.  Modulation of T-cell signalling by non-esterified fatty acids. , 2007, Prostaglandins, leukotrienes, and essential fatty acids.

[16]  L. McReynolds,et al.  Carboxy-terminal sequence divergence and processing of the polyprotein antigen from Dirofilaria immitis. , 1996, Molecular and biochemical parasitology.

[17]  A. Garofalo,et al.  The FAR proteins of filarial nematodes: secretion, glycosylation and lipid binding characteristics. , 2002, Molecular and biochemical parasitology.

[18]  A. Cooper,et al.  Novel classes of fatty acid and retinol binding protein from nematodes , 1999, Molecular and Cellular Biochemistry.

[19]  D. McManus,et al.  How helminth lipid-binding proteins offload their ligands to membranes: differential mechanisms of fatty acid transfer by the ABA-1 polyprotein allergen and Ov-FAR-1 proteins of nematodes and Sj-FABPc of schistosomes. , 2002, Biochemistry.

[20]  N. C. Price,et al.  The DvA-1 Polyprotein of the Parasitic Nematode Dictyocaulus viviparus , 1995, The Journal of Biological Chemistry.

[21]  J. Moore,et al.  The ABA-1 allergen of Ascaris lumbricoides: sequence polymorphism, stage and tissue-specific expression, lipid binding function, and protein biophysical properties , 2000, Parasitology.

[22]  L. Peelman,et al.  Cloning and sequencing of an excretory/secretory antigen from Ostertagia ostertagi fourth-stage larvae containing multiple tandem repeats. , 1995, Molecular and biochemical parasitology.

[23]  M. Kennedy,et al.  The polyprotein lipid binding proteins of nematodes. , 2000, Biochimica et biophysica acta.

[24]  H. Simpson,et al.  The failure of Haemonchus contortus excretory/secretory products to stimulate gastrin secretion in vitro , 2004, Parasitology Research.

[25]  K. Gevaert,et al.  Vaccination with an Ostertagia ostertagi Polyprotein Allergen Protects Calves against Homologous Challenge Infection , 2004, Infection and Immunity.

[26]  Joshua M. Stuart,et al.  Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans , 2002, Nature.

[27]  J. T. Jones,et al.  A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern. , 2001, The Biochemical journal.

[28]  M. Kennedy,et al.  Stage-specific secreted antigens of the parasitic larval stages of the nematode Ascaris. , 1986, Immunology.

[29]  Malcolm W. Kennedy,et al.  The FAR proteins of parasitic nematodes: their possible involvement in the pathogenesis of infection and the use of Caenorhabditis elegans as a model system to evaluate their function. , 2003, Medical Microbiology and Immunology.

[30]  W. Harnett,et al.  Structurally novel lipid-binding proteins. , 2001 .

[31]  G. Albers,et al.  Serial passage of Haemonchus contortus in resistant and susceptible sheep. , 1988, Veterinary parasitology.

[32]  D. Rathore,et al.  Identification of a 66 kDa Haemonchus contortus excretory/secretory antigen that inhibits host monocytes. , 2006, Veterinary parasitology.

[33]  B. Lentz,et al.  A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex. , 2008, Blood.

[34]  N. C. Price,et al.  The ABA-1 allergen of the parasitic nematode Ascaris suum: fatty acid and retinoid binding function and structural characterization. , 1995, Biochemistry.

[35]  M. Robinson,et al.  Proteomic analysis of the excretory‐secretory proteins of the Trichinella spiralis L1 larva, a nematode parasite of skeletal muscle , 2005, Proteomics.

[36]  A. Heck,et al.  Comprehensive Analysis of the Secreted Proteins of the Parasite Haemonchus contortus Reveals Extensive Sequence Variation and Differential Immune Recognition* , 2003, The Journal of Biological Chemistry.

[37]  M. Blaxter,et al.  Caenorhabditis elegans is a nematode. , 1998, Science.

[38]  V. Reinke,et al.  Genome-wide analysis of developmental and sex-regulated gene expression profiles in Caenorhabditis elegans. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[39]  R. Maizels,et al.  cDNA clones of Onchocerca volvulus low molecular weight antigens provide immunologically specific diagnostic probes. , 1991, Molecular and biochemical parasitology.

[40]  P. Hotez,et al.  Ac-FAR-1, a 20 kDa fatty acid- and retinol-binding protein secreted by adult Ancylostoma caninum hookworms: gene transcription pattern, ligand binding properties and structural characterisation. , 2003, Molecular and biochemical parasitology.

[41]  N. C. Price,et al.  The Ov20 Protein of the Parasitic Nematode Onchocerca volvulus , 1997, The Journal of Biological Chemistry.

[42]  M. Haswell-Elkins,et al.  Homology and heterology between the secreted antigens of the parasitic larval stages of Ascaris lumbricoides and Ascaris suum. , 1987, Clinical and experimental immunology.

[43]  G. Berx,et al.  Identification of excretory-secretory products of larval and adult Ostertagia ostertagi by immunoscreening of cDNA libraries. , 2003, Molecular and biochemical parasitology.