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Molecular cloning of a serine proteinase inhibitor from Brugia malayi.

ABSTRACT: The antigens produced by the infective-stage larvae of filarial parasites are potentially important targets for a protective immune response. A major impediment to studies on the biochemistry and molecular biology of antigens from infective larvae is a lack of parasite material. By employing a reverse transcription PCR-based strategy which exploited the presence of a conserved 22-nucleotide spliced leader sequence present at the 5' end of a proportion of nematode transcripts, spliced leader-containing cDNAs were amplified from the late-vector-stage larvae of the filarial nematode Brugia malayi. A major 1.4-kb PCR product was cloned into pBluescript. One of the PCR cDNA clones (BmY8) contained a 1,287-bp insert that encoded the first member of the serine proteinase inhibitor (serpin) superfamily to be described from nematodes. Reverse transcription PCR analysis of RNA isolated from different developmental stages of the parasite showed that transcription of the B. malayi serpin (Bmserpin) begins between days 8 and 9 of larval development within the insect vector and continues through to the adult and microfilarial stages. In immunoblot analyses of B. malayi somatic extracts, the native protein was estimated to have a molecular weight of 44,000. In immunoblots using excretory-secretory products from infective- and fourth-stage larvae, a single band with an estimated molecular weight of 75,000 was detected. A quantitative analysis of somatic extracts demonstrated that infective-stage larvae contained 10- to 16-fold-more Bmserpin than adults or microfilariae. Bmserpin was immunogenic in gerbils and was recognized strongly by sera from immunized animals. Bmserpin, which has the potential for modifying host defense responses, may play an important role in parasite survival during the early phase of vertebrate-stage development.

SUBMITTER: Yenbutr P

PROVIDER: S-EPMC173219 | biostudies-other | 1995 May

REPOSITORIES: biostudies-other

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Molecular cloning of a serine proteinase inhibitor from Brugia malayi.

Yenbutr P P Scott A L AL

Infection and immunity 19950501 5

The antigens produced by the infective-stage larvae of filarial parasites are potentially important targets for a protective immune response. A major impediment to studies on the biochemistry and molecular biology of antigens from infective larvae is a lack of parasite material. By employing a reverse transcription PCR-based strategy which exploited the presence of a conserved 22-nucleotide spliced leader sequence present at the 5' end of a proportion of nematode transcripts, spliced leader-cont ...[more]

PMID: 7729881

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Project description:BACKGROUND:Filarial nematodes, including Brugia malayi, the causative agent of lymphatic filariasis, undergo molting in both arthropod and mammalian hosts to complete their life cycles. An understanding of how these parasites cross developmental checkpoints may reveal potential targets for intervention. Pharmacological evidence suggests that ecdysteroids play a role in parasitic nematode molting and fertility although their specific function remains unknown. In insects, ecdysone triggers molting through the activation of the ecdysone receptor: a heterodimer of EcR (ecdysone receptor) and USP (Ultraspiracle). METHODS AND FINDINGS:We report the cloning and characterization of a B. malayi EcR homologue (Bma-EcR). Bma-EcR dimerizes with insect and nematode USP/RXRs and binds to DNA encoding a canonical ecdysone response element (EcRE). In support of the existence of an active ecdysone receptor in Brugia we also cloned a Brugia rxr (retinoid X receptor) homolog (Bma-RXR) and demonstrate that Bma-EcR and Bma-RXR interact to form an active heterodimer using a mammalian two-hybrid activation assay. The Bma-EcR ligand-binding domain (LBD) exhibits ligand-dependent transactivation via a GAL4 fusion protein combined with a chimeric RXR in mammalian cells treated with Ponasterone-A or a synthetic ecdysone agonist. Furthermore, we demonstrate specific up-regulation of reporter gene activity in transgenic B. malayi embryos transfected with a luciferase construct controlled by an EcRE engineered in a B. malayi promoter, in the presence of 20-hydroxy-ecdysone. CONCLUSIONS:Our study identifies and characterizes the two components (Bma-EcR and Bma-RXR) necessary for constituting a functional ecdysteroid receptor in B. malayi. Importantly, the ligand binding domain of BmaEcR is shown to be capable of responding to ecdysteroid ligands, and conversely, ecdysteroids can activate transcription of genes downstream of an EcRE in live B. malayi embryos. These results together confirm that an ecdysone signaling system operates in B. malayi and strongly suggest that Bma-EcR plays a central role in it. Furthermore, our study proposes that existing compounds targeting the insect ecdysone signaling pathway should be considered as potential pharmacological agents against filarial parasites.

Dataset Information

Molecular cloning of a serine proteinase inhibitor from Brugia malayi.

Publications

Molecular cloning of a serine proteinase inhibitor from Brugia malayi.

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