Molecular cloning and characterization of a novel immunoreactive ATPase/RNA helicase in human filarial parasite Brugia malayi.
DEAD box proteins are putative RNA unwinding proteins found in organisms ranging from mammals to bacteria. We have identified a novel immunodominant cDNA clone, BmL3-helicase, encoding DEAD box RNA helicase by immunoscreening of a larval stage cDNA library of Brugia malayi. The cDNA sequence exhibited strong sequence homology to Caenorhabditis elegans and C. briggsae RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. The clone also showed similarity with RNA helicase of Wolbachia, an endosymbiotic bacterium of filarial parasite. It was overexpressed as approximately 50 kDa His-tag fusion protein, and ATP hydrolysis assay of recombinant enzyme showed that either ATP or dATP was required for the unwinding activity, indicating BmL3-helicase as an ATP/dATP-dependent RNA helicase. The recombinant protein also demonstrated cross-seroreactivity with human bancroftian sera. The presence of BmL3-helicase in various life stages of B. malayi was confirmed by immunoblotting of parasite-life-cycle extracts with polyclonal sera against the BmL3-helicase, which showed high levels of expression in microfilaria, L(3,) and adult (both male and female) stages. In the absence of an effective macrofilaricidal agent and validated anti-filarial drug targets, RNA helicases could be utilized as a rational drug target for developing agents against the human filarial parasite.
Singh
Meghna
M
Division of Parasitology, Central Drug Research Institute, P.O. Box-173, Lucknow 226001, India.
Srivastava
K K
KK
Bhattacharya
Shailja M
SM
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D017178:Brugia malayi / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000276:immunology; D003001:Cloning, Molecular; D004605:Elephantiasis, Filarial / Q000276:immunology* / Q000469:parasitology; D005260:Female; D015723:Gene Library; D006801:Humans; D016056:Immunodominant Epitopes; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008842:Microfilariae / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D000909:Antibodies, Helminth; D016056:Immunodominant Epitopes; D000251:Adenosine Triphosphatases; D020365:RNA Helicases