Molecular characterization of a Dirofilaria immitis cDNA encoding a highly immunoreactive antigen

doi:10.1016/0166-6851(92)90094-Z

Molecular and Biochemical Parasitology

Volume 54, Issue 1, August 1992, Pages 51-62

https://doi.org/10.1016/0166-6851(92)90094-Z Get rights and content

Abstract

Dirofilaria immitis, a filarial nematode, is the causative agent of canine and feline heartworm disease. Previous research has demonstrated that immunity to D. immitis can be induced in dogs by repeated chemical abbreviation of infections while the parasite is a fourth-stage larva. Sera obtained from dogs immunized in this manner has been effective in passively transferring larval killing and stunting. These immune sera, by comparison to nonimmune sera from infected cohorts, recognize a number of unique D. immitis antigens, some of which are larval specific. In this study immune dog sera were used to screen a D. immitis larval cDNA expression library. Three overlapping cDNA clones, Di22, Di18 and Di16, were obtained that encode a portion of a large molecule, > 150 kDa, that is composed of multiples of a 399-bp repeat. This protein when immunoblotted with antibody against a recombinant expressed Di22 fusion protein is found in larval as well as adult extracts and excretory-secretory products, and is seen as a series of ascending subunits, each approximately 15 kDa larger than the previous one. This antigen is highly immunogenic, as evidenced by the strong reactivity of the recombinant expressed Di22 fusion protein with sera from immune dogs, microfilaremic dogs and infected amicrofilaremic dogs. While the function of this antigen is unknown it has significant sequence similarity with an allergen found in Ascaris.

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Molecular cloning and characterization of a nematode polyprotein antigen/allergen from the human and animal hookworm Ancylostoma ceylanicum
2014, Molecular and Biochemical Parasitology
Citation Excerpt :
However, the individual subunits have an α-helix rich structure, making them structurally different from the lipid transporters found in vertebrates. These smaller subunits are processed and secreted from worms into the host and surrounding environment [26–31]. Due to the requirement of exogenous fatty acids by the parasites, host fatty acid levels may influence pathogenesis of disease caused by parasitic nematodes.
Nematodes are unable to synthesize fatty acids de novo and must acquire them from the environment or host. It is hypothesized that two unique classes of fatty acid and retinol binding proteins that nematodes produce (fatty acid and retinol binding (FAR) and nematode polyprotein antigen/allergen (NPA)) are used to meet this need. A partial cDNA has been cloned corresponding to four subunits of a putative Ancylostoma ceylanicum NPA (AceNPA). The translated amino acid sequence of AceNPA shares sequence identity with similar proteins from Dictyocaulus viviparus, Ascaris suum, and Ostertagia ostertagi. Immunoblot experiments using a polyclonal anti-AceNPA IgG revealed proteins corresponding to the expected sizes of single, as well as two or three un-cleaved NPA subunits in adult excretory/secretory proteins and soluble adult worm extracts. Immunohistochemistry experiments localize AceNPA to the cuticle, pseudocoelomic space and testes suggesting a role in hookworm biology that is distinct from what has previously been defined for other hookworm lipid binding proteins. A single recombinant subunit of AceNPA (rAceNPAb) demonstrated binding in vitro to fluorescent fatty acids DAUDA, cis-parinaric acid, as well as retinol, at equilibrium dissociation constants in the low micromolar range. Further, in vitro data reveal that rAceNPAb binds fatty acids with chain lengths of C12–C22, with the greatest affinities for arachidonic, linoleic (C18), and eicosapentaenoic (C20) acids.
Ascaris - Antigens, Allergens, Immunogenetics, Protein Structures
2013, Ascaris: The Neglected Parasite
In comparison with other helminths that afflict humans on a global scale, Ascaris lumbricoides and ascariasis are relatively under-researched in terms of the proteins and other products to which the parasites expose their hosts. The reputation of the parasite as a source of potent allergens, and the recognition that immune hypersensitivity in the lung caused by migrating larvae is a significant problem, has served to focus attention on immune responses to Ascaris. Research has increasingly focused on the parasite’s products and responses to them in experimental rodents, and in pigs as permissive hosts for the full life-cycle of Ascaris suum. In parallel, immunoepidemiology of ascariasis in humans has been developing, often aimed at understanding how the infection polarizes immune responses. This review deals with the antigens of the parasite, how the immune repertoire to them varies dramatically between infected humans, the genetic loci that are probably involved, and its unpredictability. New work on the molecular structure of the unusual proteins produced by Ascaris and other nematodes is also covered, including the well-known ABA-1 allergen. Emphasis is also placed on a poorly understood aspect of Ascaris immunobiology, prompted by a few tantalizing observations, namely the potential diversity of surface and secreted antigens of the human parasite, and how such polymorphisms could be crucial to understanding the chronicity of ascariasis in the face of continuous loss and recruitment of the parasites themselves.
The complexity of the secreted NPA and FAR lipid-binding protein families of Haemonchus contortus revealed by an iterative proteomics-bioinformatics approach
2009, Molecular and Biochemical Parasitology
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.
The nematode polyprotein allergens/antigens
2000, Parasitology Today
Citation Excerpt :
NPAs appear to be secreted by several species of ascarid, although not by the larvae of T. canis23,24. NPAs are less prominent as somatic components in other groups of nematodes, but they have attracted attention as the surface-proximal and secreted ‘ladder’ proteins of filarial nematodes, originally revealed by radiolabelling the surface of living Brugia and Dirofilaria worms25–29. Immunoelectronmicroscopy in Brugia has indicated that its NPA (gp15/400) is present in the matrix of the basal laminae separating the hypodermal cord and the somatic musculature, between individual muscle blocks, in the basal lamina surrounding the oesophagus and at low levels within cells.
Interest in the nematode polyprotein allergens/antigens (NPAs) originally arose because they were often found to be immunodominant antigens of nematode parasites, and in some cases also potent allergens. Quite separately, they attracted attention as the ‘ladder’ proteins found close to the surface of filarial nematodes. Screening of cDNA expression libraries with antibody from infected humans or domestic animals continues to reveal more NPAs. The search for the biochemical function(s) of NPAs was originally hampered by the lack of amino acid sequence similarities between NPAs and proteins of known function, but much is now known about their biochemical activity, the highly unusual means of their biosynthesis and their gene structure. Here, Malcolm Kennedy provides an update on these intriguing proteins.
The polyprotein lipid binding proteins of nematodes
2000, Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
The nematode polyprotein allergens/antigens (NPAs) are specific to nematodes, and are synthesised as tandemly repetitive polypeptides comprising 10 or more repeated units. The polyproteins are post-translationally cleaved at consensus sites to yield multiple copies of the approximately 15-kDa NPA units. These units can be highly diverse in their amino acid sequences, but absolutely conserved signature amino acid positions are identifiable. NPA units are helix-rich and possibly fold as four helix bundle proteins. The NPA units have relatively non-specific lipid binding activities, binding fatty acids and retinoids, with dissociation constants similar to those of lipid transport proteins of vertebrates. Fluorescence-based analysis has indicated that, like most lipid transport proteins, the ligand is taken into the binding site in its entirety, but the binding site environment is unusual. NPAs are synthesised in the gut of nematodes, and presumably act to distribute small lipids from the gut, via the pseudocoelomic fluid, to consuming tissues (muscles, gonads, etc.). In some species, one of the units has a histidine-rich extension peptide which binds haems and certain divalent metal ions. NPAs appear to be released by parasitic nematodes, and may thereby be involved in modification of the local inflammatory and immunological environment of the tissues they inhabit by delivering or sequestering pharmacologically active lipids – they are known to bind arachidonic acids and some of its metabolites, lysophospholipids, and retinoids. NPAs are the only known lipid binding protein made as polyproteins, and are exceptions to the rule that repetitive polyproteins are only produced by cells undergoing programmed cell death and producing specialist products.
Carboxy-terminal sequence divergence and processing of the polyprotein antigen from Dirofilaria immitis
1996, Molecular and Biochemical Parasitology
A polyprotein composed of multiple units arranged in direct tandem arrays has been identified in parasitic and free living nematodes. Analysis of previously cloned units from the Dirofilaria immitis polyprotein antigen (DiPA) indicated the units were nearly identical but here we demonstrate that they segregate into two related families. The consensus repeats, DiPA-CR1 and CR2, derived for each family are 80% identical. However, the repeats at the C-terminus of the polyprotein have diverged from DiPA-CR1 and CR2. This was shown by DNA sequence and Southern blot analysis of a 1.9 kb cDNA clone that encodes 4.4 C-terminal repeats (DiPA-TR1 through TR5). DiPA-TR3 through TR5 show 27–52% amino acid identity with the consensus repeats and 31–35% amino acid identity with one another. Metabolic labeling studies have shown that cleavage of DiPA generates a protein ‘ladder’ from 14 to > 200 kDa. RRKR, a cleavage motif of subtilisin-like proprotein convertases, was identified as the natural cleavage site. In vitro digestion experiments with proteinase K suggest a structural model for DiPA consisting of protease resistant cores joined by protease sensitive linkers containing the RRKR site. This motif is absent between DiPA-TR3 and TR4 and has been altered to KR between DiPA-TR4 and TR5. An immunoblot of D. immitis extract probed with anti-DiPA-TR4/5 serum demonstrates the absence of cleavage at these sites. These divergent repeats provide an opportunity to investigate processing of the D. immitis polyprotein in vivo.

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^∗: Present address: Department of Molecular Biology, DNAX Research Institute, Palo Alto, CA

^∗∗: Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA

^∗∗∗: Department of Infectious Disease, Roche Molecular Systems, Emeryville, CA; USA.

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Molecular characterization of a Dirofilaria immitis cDNA encoding a highly immunoreactive antigen

Abstract

Exp. Parasitol.

J. Biol. Chem.

Gene

J. Mol. Biol.

Induction of protective immunity in dogs to infection with Dirofilaria immitis using chemically-abbreviated infections

Am. J. Trop. Med. Hyg.

Passive transfer of protective immunity to larval Dirofilaria immitis from dogs to BALB/c mice

J. Parasitol.

Identification of Dirofilaria immitis larval antigens with immunoprophylactic potential using sera from immune dogs

J. Immunol.

Clinicopathologic and histologic evaluation of Dirofilaria immitis-induced nephropathy in dogs

Am. J. of Trop. Med. Hyg.

Indirect fluorescence antibody test in occult dirofilariasis

Am. J. Vet. Res.

N-terminal amino acid sequence identity between a major allergen of Ascaris lumbricoides and Ascaris suum, and MHC-restricted IgE responses to it

Immunology

In vitro culture of Dirofilaria immitis third- and fourth-stage larvae under defined conditions

J. Parasitol.

Parasite excretory-secretory antigen and antibody to excretory-secretory antigen in body fluids and kidney tissue of Dirofilaria immitis infected dogs

Am. J. Trop. Med. Hyg.

Metabolic labeling of Dirofilaria immitis third- and fourth-stage larvae and their excretory-secretory products

J. Parasitol.

Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction

Anal. Biochem.

One-tube double-stranded cDNA synthesis using cloned M-MLV reverse transcriptase

Focus

Molecular Cloning