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Expression and characterisation of asparagine‐tRNA synthetase: the key enzyme that mediates angiogenesis in lymphatic filariasis (585.10)
Author(s) -
Dhanasekaran Jeeva Jothi,
Solaiyappan Shanmugam,
D Dhanraj Muthu,
Chatterjee Suvro,
Kron Michael,
Dhanasekaran Anuradha
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.585.10
Subject(s) - angiogenesis , brugia malayi , biology , microbiology and biotechnology , mtt assay , enzyme , recombinant dna , biochemistry , in vitro , filariasis , cancer research , immunology , gene , helminths
Asparagine‐tRNA synthetase is an enzyme which belongs to the class‐IIb aminoacyl‐tRNA synthetases(AARSs) an heterogeneous family of enzymes,that play a role in protein biosynthesis. Brugi Malayi parasites secrete this 63kDa high molecular weight enzyme. It has not been documented clearly,how humans infected with filariasis have lymphatic dysfunction. We have cloned and purified the AsnRs enzyme to evaluate the role played by this enzyme in angiogenesis in Filarial patients.The recombinant plasmid of pET‐15B encoding Brugia malayi Asparagine‐tRNA synthetase was transformed into the competent cells of E.coli BL21(DE3),induced to express by IPTG,purified by Immobilized metal ion affinity chromatography and identified by western blotting. Purified Recombinant Brugia malayi AsnRS was used to perform cellular based assays using endothelial cells‐ MTT assay,Chemotactic assay,ring formation assay and tube formation assay in EAhy926 cells.We also carried out angiogenesis assays in chick chorioallantoic membrane (CAM) models. Recombinant AsnRS induces proliferation in MTT assay,migration in chemotaxis assay,formation of endothelial ring structure in ring formation assay and tubular structures in matric gel assay. Additionally,BmAsnRs promotes the formation and widening of blood vessel mediated angiogenesis in CAM model. Our results showed the enzyme BmAsnRs to be a potent pro‐angiogenic mediator in vitro and CAM models. These results throw light on the possibility of AsnRs as a drug target for the control of Filariasis and thus this enzyme can be targeted for the control of angiogenesis‐associated pathology in Filariasis.