Abstract
BackgroundMethyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. However, the role of MTFs in immune mechanism during host–parasite interaction has not been addressed yet.ResultsAn open reading frame (764 bp) of methyltransferase-type 12 gene of H. contortus denoted as HcMTF-12, was successfully cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by prokaryotic expression in Escherichia coli BL21 (DE3 strain). The recombinant HcMTF-12 protein (rHcMTF-12) was about 47 kDa along with a fusion vector protein of 18 kDa. Immunoblot results identified the native protein MTF-12 with antibodies produced in rats against rHcMT-12, whereas rHcMTF-12 protein was recognized with sera of goat experimentally infected with H. contortus. Immunohistochemical analysis revealed that the native MTF-12 protein was mainly located in the periphery (cuticle) of parasite sections as well as within the pharynx and intestinal region. An immunofluorescence assay validated that rHcMTF-12 attached to the surface of goat PBMCs. Furthermore, the cytokines transcription of IL-2, IFN-γ and IL-4 transcripts of PBMCs incubated with rHcMTF-12 were enhanced in a dose-dependent manner. The secretion of TGF-β1 and IL-10 was significantly decreased. However, IL-6 production was not significantly different as compared to the control groups. Moreover, the migration activity and nitric oxide (NO) production by PBMCs were induced considerably, whereas the proliferation of PBMCs cells was negatively affected when incubated with the rHcMTF-12 protein.ConclusionsOur findings suggest that HcMTF-12 significantly mediated the functions of PBMCs, and it might be a potential candidate for therapeutic interventions against haemonchosis.
Highlights
Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals
Amplification and cloning of the HcMTF‐12 gene Amplification of the HcMTF-12 gene was successfully performed by PCR from H. contortus cDNA with specific primer pairs with the fragment detected at the desired size of 764 bp (Fig. 1)
The target sequence was further analyzed by multiple alignment with a number of published sequences and determined that the cloned ORF of HcMTF-12 belongs to the H. contortus Adenosyl-methionine superfamily
Summary
Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. Methyltransferases (MTFs) are a broad range of enzymes, which are ubiquitously expressed in varied organisms ranging from bacteria to animals [1]. MTFs are membrane-bound enzymes that transfer a methyl group from S-adenosyl-l-methionine to thiol, amino or catechol acceptor molecules and this reaction modifies RNA, DNA and protein molecules for regulatory activities [2, 3]. MTFs have been implicated in transcriptional regulation, subcellular protein and RNA localization, DNA-damage repair and signal transduction [5]. A recent research in the context of drug discovery and drug development revealed that MTFs are involved in using of S-Adenosyl methionine (SAM) analogs to methylate naturally produced anticancer agents which keeps alternate alkyl group as a substitute for methyl [8]
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