Regulated use of an alternative spliced leader exon in the plant parasitic nematode Meloidogyne javanica.
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Alternative Splicing↗
Biomedical subjects
M L Blaxter
Publications and source records attributed to M L Blaxter.
The 5S ribosomal RNA intergenic region of parasitic nematodes: variation in size and presence of SL1 RNA.
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Animals↗
Genes expressed in Brugia malayi infective third stage larvae.
We have used a tag sequencing approach to survey genes expressed in the third stage infective larvae of the human filarial nematode parasite Brugia malayi. RNA was isolated from late vector-stage L3 larvae after days 9 or 10 of infection in mosquitos, and converted to cDNA by reverse transcriptase. Double-stranded cDNA was produced by either conventional methods (non-SL cDNA library) or by PCR using the nematode spliced leader (SLI) and oligo(dT) primers (SL cDNA library). Two clone libraries (one from SL and one from non-SL cDNAs) were constructed in lambda ZapII. A set of these full-length clones was selected and 596 inserts were sequenced from the 5' end. We have identified 364 B. malayi genes (the majority of which are new) that encode housekeeping proteins, structural proteins, proteins of immediate immunological or drug-discovery interest as well as a large class of novel sequences which may prove to have significant involvement in host invasion. Extensive, genome-wide approaches to the analysis of larval gene expression are now possible for B. malayi. We present several examples of this approach.
Amino Acid Sequence↗
Isolation, characterization and immunolocalization of a globin-like antigen from Ostertagia ostertagi adults.
Western blot analysis using an anti-globin rabbit serum Rb94 revealed a major band of 17 kDa in extracts of Ostertagia ostertagi adults and 4th-stage larvae. The adult stage globin-like antigen (OoAdGlb) was purified from total worm extracts by liquid chromatography. The protein has an estimated molecular mass of 36 kDa under non-reducing conditions, suggesting a dimeric structure containing 2 non-covalently linked 17 kDa monomers. Tryptic peptides were sequenced and showed strong similarities with the globins of free-living and parasitic nematodes. Immunolocalization revealed the presence of this globin-like antigen in the body wall musculature and/or the cuticle of O. ostertagi adults. An enzyme-linked immunosorbent assay based on the purified OoAdGlb showed no differences in response between calves infected by O. ostertagi and/or Cooperia oncophora and the negative controls.
Amino Acid Sequence↗
An abundant, trans-spliced mRNA from Toxocara canis infective larvae encodes a 26-kDa protein with homology to phosphatidylethanolamine-binding proteins.
A full-length mRNA encoding a secreted 26-kDa antigen of infective larvae of the ascarid nematode parasite Toxocara canis has been identified. This was characterized as a 1,082-base pair clone highly abundant (0.8-1.9%) in cDNA prepared from infective stage larvae but absent from cDNA from adult male worms. Sequence analysis revealed an open reading frame corresponding to a hydrophilic 263-amino acid residue polypeptide with a 20-residue N-terminal signal peptide, indicating that it is secreted. The 5' end of the cDNA was isolated by polymerase chain reaction using a primer containing the nematode-spliced leader sequence, SL1, showing that the mRNA is trans-spliced. The molecular mass of the putative protein with the signal peptide removed is 26.01 kDa, and antibody to the recombinant protein expressed in bacterial vectors reacts with a similarly sized protein in T. canis excretory/secretory (TES) products. An identical sequence was obtained from a genomic clone isolated by expression screening with mouse antibody to TES. The 72 amino acid residues adjacent to the signal peptide form two homologous 36-residue motifs containing 6 cysteine residues; this motif is found also in the T. canis-secreted glycoprotein TES-120 and in genes of Caenorhabditis elegans. Sequence data base searches revealed significant similarity to 7 other sequences in a newly recognized gene family of phosphatidylethanolamine-binding proteins that includes yeast, Drosophila, rat, bovine, simian, and human genes and a representative from the filarial nematode Onchocerca volvulus. Assays with the T. canis recombinant 26-kDa protein expressed as a fusion with maltose-binding protein have confirmed phosphatidylethanolamine-binding specificity for this novel product.
Amino Acid Sequence↗
Characterisation of an immunodominant glycoprotein antigen of Onchocerca volvulus with homologues in other filarial nematodes and Caenorhabditis elegans.
The full-length cDNA corresponding to an Onchocerca volvulus antigen, OvMBP/11, which had been selected as a serodiagnostic tool was isolated, sequenced, and the native antigen encoded by the cDNA characterised. The cDNA encodes a protein of 20.5 kDa (termed Ov 20) containing a putative signal peptide. Southern blot analysis indicates that there is only a single O. volvulus gene corresponding to Ov 20 but it has significant sequence similarity to genes corresponding to two 20.5-kDa predicted proteins of Caenorhabditis elegans. Homologues of the Ov 20 gene were detected at high stringency by Southern blot in the other Onchocerca species O. gibsoni, and O. gutturosa and at lower stringency in the related filarial nematodes Brugia malayi and Acanthocheilonema viteae. The Ov 20 native antigen has two molecular mass forms, 20 and 22 kDa, in all the life cycle stages studied. These isoforms have different levels of N-linked glycosylation on a peptide backbone of 17.5 kDa. Immunolocalisation and in situ hybridisation studies demonstrated that Ov 20 is transcribed and translated in the body wall of adult females and also in microfilariae, third and fourth stage larvae. Antigen was detected in the supernatant of in vitro cultured adult female nematodes. The B. malayi and A. viteae homologues are antigenically cross-reactive to Ov 20, share the same size peptide backbone but differ in their degree of glycosylation.
Amino Acid Sequence↗
Structural characterization of an Ascaris myoglobin.
Globin was purified from the body wall of adults of the parasitic nematode Ascaris suum. Internal peptide fragments were sequenced and cDNAs encoding a polypeptide of 154 amino acids isolated by polymerase chain reaction. The polypeptide lacks a signal sequence, identifying it as a cytosolic myoglobin-like species. The native protein is a dimer. The predicted amino acid sequence shares several unusual substitutions with other nematode globins. Like the abundant pseudocoelomic A. suum hemoglobin it has a Tyr at B10 and a Gln at E7, substitutions thought to be determinants of high affinity. However, the 10-fold lower oxygen affinity of body wall globin suggests that in this molecule Tyr(B10) does not form an additional hydrogen bond with the heme bound oxygen. Evolutionary analysis of the nematode globins suggests that the monodomain myoglobin-like molecules and the two-domain hemoglobin-like molecules diverged about 500 million years ago, well before the divergence of the ascarid genera Ascaris and Pseudoterranova. The absence of introns in the A. suum myoglobin, in contrast to other nematode globin genes, is consistent with the hypothesis that during evolution intron elimination was the predominant event.
Amino Acid Sequence↗
Molecular genealogy of some nematode taxa as based on cytochrome c and globin amino acid sequences.
We have begun to reconstruct the ancient history of the nematode phylum based on cytochrome c and globin amino acid sequences. The data suggest that the nematode ancestor diverged from a line leading to mammals about 1 billion years ago and that the most recent common ancestor of the extant species Caenorhabditis elegans, Trichostrongylus colubriformis, Nippostrongylus brasiliensis, Ascaris suum, and Pseudoterranova decipiens lived about 550 MY ago. The rhabditids and strongylids emerged as one offshoot of this ancestor, the ascarids as another. Rhabditids and strongylids diverged some 400 MY ago, whereas the genera Trichostrongylus and Nippostrongylus diverged slightly over 200 MY ago. A gene duplication event in the strongylid branch is predicted to have occurred around 250-335 MY ago. There are two globin genes in Nippostrongylus, expressed in anatomically distinct compartments (body and cuticle), and the single sequence from Trichostrongylus is most like the Nippostrongylus body globin gene. A strikingly different duplication event occurred within the same period in the line leading to the extant ascarid genera, creating a single polypeptide containing two globin domains. The genera Ascaris and Pseudoterranova diverged some 150-250 MY ago. Interestingly, the second globin repeat evolved at a faster rate in both species examined. This is possibly related to the acquisition of an unusual carboxyterminal extension, composed of alternating positively and negatively charged residues, that is necessary for the assembly of several monomers into the native polymeric molecules.
Amino Acid Sequence↗
Sequence, expression and evolution of the globins of the parasitic nematode Nippostrongylus brasiliensis.
The globins of the nematode parasite Nippostrongylus brasiliensis have oxygen affinities 100-fold higher than the rodent host's haemoglobins. Two isoforms are found, one located in the cuticle, and the other in the body of the nematode. Both isoforms have been cloned and analysed for clues as to function and evolution. The body globin isoform is first expressed upon invasion of the mammalian host. The abundant cuticular globin is expressed only by adult nematodes in the gut, and differs significantly from the body globin. Both globins are found as trans-spliced mRNAs: the developmental pattern of expression of the mRNA parallels the protein expression. The pattern of the nematode globin genes is complex. Comparison with other nematode globin sequences suggests that N. brasiliensis is more closely related to Caenorhabditis elegans than to ascarid species. At least two gene duplication events are predicted: gene duplication preceded the radiation of the important vertebrate-parasitic strongylid nematode species. Both N. brasiliensis globins have a central intron the exact position of which suggests that it arose from an independent insertion event in the strongylid-rhabditid line. The globins have been expressed in Escherichia coli as functional holenzymes as a prelude to studies to elucidate the origin of their extraordinary oxygen affinity.
Amino Acid Sequence↗
Cuticle surface proteins of wild type and mutant Caenorhabditis elegans.
The molecular components of the surface of the free-living nematode Caenorhabditis elegans have been identified by surface-specific radioiodination. Four compartments were defined by fractionation of labeled wild type (N2 strain) adult hermaphrodites. Organic solvents extracted cuticular lipids. Homogenization in detergents released a single, non-collagenous, hydrophobic protein. This is not glycosylated and is a heterodimer of 6.5- and 12-kDa subunits. The third compartment, proteins solubilized by reducing agents, included both the cuticular collagens and the heterodimer. Residual material corresponds to the cuticlin fraction. Larval stages showed a similar pattern, except that the dauer larva had an additional 37-kDa detergent-soluble protein. Other species of rhabditid nematodes displayed similar profiles, and comparison with parasitic species suggests that this simple pattern may be primitive in the Nematoda. A C. elegans strain mutant in cuticular collagen (rol-6) had a pattern identical to that of wild type, but another morphological mutant (dpy-3) [corrected] and several mutants that differ in surface reactivity to antibody and lectins (srf mutants) also had striking differences in surface labeling patterns.
Animals↗
Forms and functions of nematode surfaces.
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Animals↗
Nemoglobins: divergent nematode globins.
Globins are proteins commonly associated with oxygen transport in vertebrate blood, but the invertebrate phyla display a wide variety of globin types that reflect their disparate life styles and evolutionary history. It has been known for over 100 years that parasitic nematodes contain globins, but recent molecular investigations are only now beginning to shed some light on their curious properties. Mark Blaxter here describes the diversity of the different globins found in nematodes, and reviews emerging data on their evolution and function.
Journal Article↗
Identification of the major soluble cuticular glycoprotein of lymphatic filarial nematode parasites (gp29) as a secretory homolog of glutathione peroxidase.
We have cloned and identified the major cuticular glycoprotein (gp29) of lymphatic filarial nematode parasites as a homolog of the antioxidant enzyme glutathione peroxidase. The derived amino acid sequence predicted a protein of 25.8 kDa, with an amino-terminal hydrophobic signal peptide and two sites for N-linked glycosylation, consistent with the documented properties of gp29. Transcription of a full-length cDNA in an SP65 vector and subsequent translation of the RNA in reticulocyte lysates in vitro generated a protein of 27 kDa, which was glycosylated upon the addition of pancreatic microsomal membranes. A postulated role for this secreted enzyme could be inhibition of the oxidative burst of leukocytes and neutralization of secondary products of lipid peroxidation, thus providing one explanation for the resistance of these parasites to immune effector mechanisms and their persistence in the mammalian host.
Amino Acid Sequence↗
Toxocara canis: a labile antigenic surface coat overlying the epicuticle of infective larvae.
An electron-dense coat covering the surface of Toxocara canis infective-stage larvae is described. This coat readily binds to cationized ferritin and ruthenium red, indicating a net negative charge and mucopolysaccharide content, and can be visualized by immuno-electron microscopy only if cryosectioning is employed. Monoclonal antibodies reactive to the surface of live larvae bind the surface coat but not the underlying cuticle in ultrathin cryosections. The surface coat is dissipated on exposure to ethanol, explaining the lack of surface reactivity of conventionally prepared immunoelectron microscopy sections of T. canis. Differential ethanol extraction of surface-iodinated larvae demonstrates that the major component associated with the coat is TES-120, a 120-kDa glycoprotein previously identified by surface iodination, which is also a dominant secreted product. The surface-labeled TES-70 glycoprotein is linked with a more hydrophobic stratum at the surface, while a prominent 32-kDa glycoprotein, TES-32, is more strongly represented within the cuticle itself. Antibody binding to the coat under physiological conditions results in the loss of the surface coat, but this process is arrested at 4 degrees C. This result gives a physical basis to earlier observations on the shedding of surface-bound antibodies by this parasite. An extracuticular surface coat has been demonstrated on Toxocara larvae prior to hatching from the egg and during all stages of in vitro culture, suggesting that it may play a role both in protecting the parasite on hatching in the gastrointestinal tract and on subsequent tissue invasion in evading host immune responses directed at surface antigens.
Animals↗
Nematode surface coats: actively evading immunity.
The classical view of nematode parasites depicts their surface as the epicuticle, the outermost layer of a thick extracellular cuticle. However, many stages and species of nematode have been found to bear an electron-dense cuter envelope distinct from and distal to the epicuticle itself. In this review, Mark Blaxter and colleagues summarize some wide-ranging studies in both free-living and parasitic nematodes, and suggest that, in many cases, it is the surface coat rather than the cuticle that displays dynamic properties thought to be involved in immune evasion by parasites.
Journal Article↗
A proline-rich structural protein of the surface sheath of larval Brugia filarial nematode parasites.
Both cDNA and genomic DNA sequences have been isolated which encode a proline-rich precursor protein of the sheath from microfilariae, the first stage larvae of the filarial nematode parasites Brugia pahangi and Brugia malayi. This 22-kDa protein is soluble only under reducing conditions and is extensively cross-linked by both disulfide and nonreducible bonds. Immunogold electron microscopy shows that the protein is localized exclusively in the sheath, a vestigial remnant of the eggshell, which is retained by and encloses the mature microfilaria. Analysis by Western blotting confirms that the protein is expressed only in microfilariae and adult female worms, although transcripts are detectable only in adult females. The deduced amino acid sequence contains a short N-terminal hydrophobic putative leader sequence, a central repetitive domain that contains 14 copies of a degenerate 5-amino acid repeat with the consensus sequence Met-Pro-Pro-Gln-Gly, and a C-terminal proline-rich domain flanked by clusters of cysteine residues. These clusters can be aligned with cysteine residues implicated in cross-linking of a family of cuticular collagens originally identified in Caenorhabditis elegans but which extends to other nematodes.
Amino Acid Sequence↗
Identification and characterisation of a Leishmania donovani antigen belonging to the 70-kDa heat-shock protein family.
A Leishmania donovani promastigote cDNA library was screened with serum obtained from a patient infected with visceral leishmaniasis. Sequence analysis of a clone obtained from this library revealed that the 600-bp insert corresponded to the carboxy-terminal region of an antigen related to the 70-kDa heat-shock protein family. The full-length sequence of the corresponding gene (1959 nucleotides) was determined after isolation of genomic clones. Genes encoding the antigen are present on a single chromosome as a series of approximately twelve 3.7-kb direct tandem repeats. The antigen can be identified as a 70-kDa heat-shock cognate protein by virtue of its molecular mass, sequence and constitutive expression during heat shock. It is expressed at all stages of the parasite life-cycle. Antibodies against the lambda gt11 fusion protein were detected in more than 50% of serum samples obtained from patients with visceral leishmaniasis, but were not detected in sera from patients with cutaneous leishmaniasis or Chagas' disease.
Amino Acid Sequence↗
Cuticular proteins of Brugia filarial parasites.
Surface-labelling techniques have been used to delineate a number of constituent molecules of the cuticle in adult stage Brugia malayi and Brugia pahangi. These molecules can be separated by virtue of their physical properties, and localised either by sequential solubilisation of intact cuticles or immunoelectron microscopy with relevant antisera. The major structural components of the cuticular matrix consist of a set of collagenous proteins of diverse molecular weight ranging from 36 to 160 kDa, cross-linked by disulphide bonds and confined to the basal and inner cortical layers. Each stage of the parasite has a distinctive set of between 12 to 25 collagenous proteins whose synthesis is regulated temporally with respect to moulting. As in other nematodes, the outer cortex and epicuticle is composed of a cross-linked insoluble proteinaceous structure. Two non-structural and water-soluble proteins are also resolved by Iodogen-mediated labelling; a 15 kDa peptide which shows no evidence of glycosylation, and a major 29 kDa glycoprotein, which carries at least two N-linked oligosaccharide chains and which we have termed Gp29. The former protein can be detected in L3, L4 and adult B. malayi by surface labelling, whereas Gp29 appears to be restricted to L4 and adult worms. The possible significance of cuticular proteins as targets of immunity or causative agents of pathology is discussed.
Animals↗