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Characterization of a platelet-activating factor acetylhydrolase secreted by the nematode parasite Nippostrongylus brasiliensis.

ABSTRACT: Nippostrongylus brasiliensis, a small nematode parasite of the gastrointestinal tract of rodents, secretes an enzyme that cleaves the proinflammatory molecule platelet-activating factor to its inactive lyso-form. The enzyme activity of Ca(2+)-dependent and does not exhibit interfacial activation. It does not require the addition of reducing agents for maximal activity, and is not inhibited by thiol-active reagents. Sensitivity of inhibitors suggests the involvement of serine and histidine residues in the enzyme activity. As described for other platelet-activating factor acetylhydrolases, it cannot cleave, nor is it inhibited by, long-chain diacyl phospholipids that are typical substrates for phospholipases A2. The purified enzyme was resolved by SDS/PAGE as a heterodimer composed of two protein subunits with apparent molecular masses of 38 and 25 kDa. The properties of the nematode enzyme thus differ from those described for the mammalian enzymes, but are more closely related to those of an acetylhydrolase than a phospholipase.

SUBMITTER: Grigg ME

PROVIDER: S-EPMC1217520 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Lentiviral transduction facilitates RNA interference in the nematode parasite Nippostrongylus brasiliensis.

Project description:Animal-parasitic nematodes have thus far been largely refractory to genetic manipulation, and methods employed to effect RNA interference (RNAi) have been ineffective or inconsistent in most cases. We describe here a new approach for genetic manipulation of Nippostrongylus brasiliensis, a widely used laboratory model of gastrointestinal nematode infection. N. brasiliensis was successfully transduced with Vesicular Stomatitis Virus glycoprotein G (VSV-G)-pseudotyped lentivirus. The virus was taken up via the nematode intestine, RNA reverse transcribed into proviral DNA, and transgene transcripts produced stably in infective larvae, which resulted in expression of the reporter protein mCherry. Improved transgene expression was achieved by incorporating the C. elegans hlh11 promoter and the tbb2 3´-UTR into viral constructs. MicroRNA-adapted short hairpin RNAs delivered in this manner were processed correctly and resulted in partial knockdown of β-tubulin isotype-1 (tbb-iso-1) and secreted acetylcholinesterase B (ache-B). The system was further refined by lentiviral delivery of double stranded RNAs, which acted as a trigger for RNAi following processing and generation of 22G-RNAs. Virus-encoded sequences were detectable in F1 eggs and third stage larvae, demonstrating that proviral DNA entered the germline and was heritable. Lentiviral transduction thus provides a new means for genetic manipulation of parasitic nematodes, including gene silencing and expression of exogenous genes.

| S-EPMC7864396 | biostudies-literature

Secreted proteomes of different developmental stages of the gastrointestinal nematode Nippostrongylus brasiliensis.

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Platelet-activating factor acetylhydrolase: A biomarker in Hymenoptera venom allergy?

Project description: Not available

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Leishmania parasites possess a platelet-activating factor acetylhydrolase important for virulence.

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| S-EPMC3492548 | biostudies-literature

Intracellular erythrocyte platelet-activating factor acetylhydrolase I inactivates aspirin in blood.

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| S-EPMC3186390 | biostudies-literature

Signal sequence analysis of expressed sequence tags from the nematode Nippostrongylus brasiliensis and the evolution of secreted proteins in parasites.

Project description:BackgroundParasitism is a highly successful mode of life and one that requires suites of gene adaptations to permit survival within a potentially hostile host. Among such adaptations is the secretion of proteins capable of modifying or manipulating the host environment. Nippostrongylus brasiliensis is a well-studied model nematode parasite of rodents, which secretes products known to modulate host immunity.ResultsTaking a genomic approach to characterize potential secreted products, we analyzed expressed sequence tag (EST) sequences for putative amino-terminal secretory signals. We sequenced ESTs from a cDNA library constructed by oligo-capping to select full-length cDNAs, as well as from conventional cDNA libraries. SignalP analysis was applied to predicted open reading frames, to identify potential signal peptides and anchors. Among 1,234 ESTs, 197 (~16%) contain predicted 5' signal sequences, with 176 classified as conventional signal peptides and 21 as signal anchors. ESTs cluster into 742 distinct genes, of which 135 (18%) bear predicted signal-sequence coding regions. Comparisons of clusters with homologs from Caenorhabditis elegans and more distantly related organisms reveal that the majority (65% at P < e-10) of signal peptide-bearing sequences from N. brasiliensis show no similarity to previously reported genes, and less than 10% align to conserved genes recorded outside the phylum Nematoda. Of all novel sequences identified, 32% contained predicted signal peptides, whereas this was the case for only 3.4% of conserved genes with sequence homologies beyond the Nematoda.ConclusionsThese results indicate that secreted proteins may be undergoing accelerated evolution, either because of relaxed functional constraints, or in response to stronger selective pressure from host immunity.

| S-EPMC463072 | biostudies-literature

C-type lectins from the nematode parasites Heligmosomoides polygyrus and Nippostrongylus brasiliensis.

Project description:The C-type lectin superfamily is highly represented in all metazoan phyla so far studied. Many members of this superfamily are important in innate immune defences against infection, while others serve key developmental and structural roles. Within the superfamily, many proteins contain multiple canonical carbohydrate-recognition domains (CRDs), together with additional non-lectin domains. In this report, we have studied two gastrointestinal nematode parasites which are widely used in experimental rodent systems, Heligmosomoides polygyrus and Nippostrongylus brasiliensis. From cDNA libraries, we have isolated 3 new C-type lectins from these species; all are single-CRD proteins with short additional N-terminal domains. The predicted Hp-CTL-1 protein contains 156 aa, Nb-CTL-1 191 aa and Nb-CTL-2 183 aa; all encode predicted signal peptides, as well as key conserved sequence motifs characteristic of the CTL superfamily. These lectins are most similar to C. elegans CLEC-48, 49 and 50, as well as to the lectin domains of mammalian immune system proteins CD23 and CD206. RT-PCR showed that these H. polygyrus and N. brasiliensis genes are primarily expressed in the gut-dwelling adult stages, although Nb-CTL-2 transcripts are also prominent in the free-living infective larval (L3) stage. Polyclonal antibodies raised to Hp-CTL-1 and Nb-CTL-1 reacted to both proteins by ELISA, and in Western blot analysis recognised a 15-kDa band in secreted proteins of adult N. brasiliensis (NES) and a 19-kDa band in H. polygyrus ES (HES). Anti-CTL-1 antibody also bound strongly to the cuticle of adult H. polygyrus. Hence, live parasites release C-type lectins homologous to some key receptors of the mammalian host immune system, raising the possibility that these products interfere in some manner with immunological recognition or effector function.

| S-EPMC2792708 | biostudies-literature

Trafficking of platelet-activating factor acetylhydrolase type II in response to oxidative stress.

Project description:Platelet-activating factor acetylhydrolase type II (PAFAH-II) is an intracellular phospholipase A(2) enzyme that hydrolyzes platelet-activating factor and oxidatively fragmented phospholipids. This N-terminally myristoylated protein becomes associated with cytoplasm-facing cell membranes under oxidative stress. The structural requirements for binding of PAFAH-II to membranes in response to oxidative stress are unknown. To begin elucidating the mechanism of trafficking and stress response, we constructed a homology model of PAFAH-II. From the predicted membrane orientation of PAFAH-II, the N-terminal myristoyl group and a hydrophobic patch are hypothesized to be involved in membrane binding. Localization studies of human PAFAH-II in HEK293 cells indicated that an unmyristoylated mutant remained cytoplasmic under stressed and unstressed conditions. The myristoylated wild-type enzyme was partially localized to the cytoplasmic membranes prior to stress and became more localized to these membranes upon stress. A triple mutation of three hydrophobic patch residues of the membrane binding region likewise did not localize to membranes following stress. These results indicate that both the myristoyl group and the hydrophobic patch are essential for proper trafficking of the enzyme to the membranes following oxidative stress. Additionally, colocalization studies using organelle-specific proteins demonstrate that PAFAH-II is transported to the membranes of both the endoplasmic reticulum and Golgi apparatus.

| S-EPMC3183284 | biostudies-literature

miRNA data from extracellular vesicles secreted by Nippostrongylus brasiliensis

Project description:miRNA-Seq analysis of the miRNA present in the extracellular vesicles secreted by the nematode Nippostrongylus brasiliensis

2018-04-01 | GSE111478 | GEO

Exploring the plasmatic platelet-activating factor acetylhydrolase activity in patients with anti-phospholipid antibodies.

Project description:PURPOSE:To explore the role of plasmatic platelet-activating factor acetylhydrolase (PAF-AH), a marker of cardiovascular risk, in patients with anti-phospholipid antibodies (aPL). METHODS:PAF-AH activity was assessed in a series of 167 unselected patients screened for aPL in a context of thrombotic events, risk of thrombosis or obstetric complications and in 77 blood donors. RESULTS:116/167 patients showed positive results for at least one aPL among IgG/IgM anti-prothrombin/phosphatidylserine (aPS/PT), anti-cardiolipin (aCL), anti-beta2-glycoprotein I (a?2GPI) or lupus anticoagulant (LAC), while 51/167 patients resulted aPL-negative. LAC+ patients disclosed higher PAF-AH than LAC-negative (22.1 ± 6.4 nmol/min/ml vs. 19.5 ± 4.1 nmol/min/ml; p = 0.0032), and aPL-negative patients (p = 0.03). Patients presenting positive IgG a?2GPI disclosed higher PAF-AH than patients with only IgM a?2GPI-positive antibodies (23.1 ± 7.2 nmol/min/ml vs. 20.1 ± 5.3 nmol/min/ml; p = 0.035), as well as than patients showing only isolated LAC, aCL or aPS/PT (16.9 ± 3.8 nmol/min/ml; p = 0.003). CONCLUSIONS:PAF-AH plasmatic activity is particularly up-regulated in LAC+ and in a?2GPI IgG+ patients, possibly representing an alternative prognostic biomarker for the therapeutic management of APS patients.

| S-EPMC5365449 | biostudies-literature

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