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Homologous sequences of cardiac iso-form of rat a2 macroglobulin Inmarine sponges (Spongia officinalis)

Author(s): M.Sivagama Sundari., 1 S.T. Somasundaram., 2 Chellam Rajamanickam*1

Journal: Journal of Pharmacy Research
ISSN 0974-6943

Volume: 4;
Issue: 3;
Start page: 589;
Date: 2011;
Original page

Keywords: Alpha 2 macroglobulin | Spongia officinalis | HT29 colon cancer cell lines | polyclonal antibodies

Marine Sponges as model system for evolutionary biology are offering key insight into the complexities of higher metazoans. The marine sponges are able to distinguish self and non self which is a central feature of immune system. The broad spectrum protease inhibitor alpha 2 macroglobulin, complement factors (C3, C4, C5) ranging from corals to humans play a vital role in host defence mechanism. There is no evidence for the presence of alpha 2 macroglobulin or its related molecule in sponges yet. PCR based analysis of the cDNA from a primitive metazoan, poriferan, a marine sponge namely spongia officinalis using the forward primer of N terminal and the reverse primer of C terminal regions resulted in an amplicon size of 738bp. Sequencing through outsourcing showed the presence of the N terminal and C terminal regions adjacently, lacking other functional domains of 182kDa (Bait, Thiol ester, Growth factor Binding Domain). Computational analysis revealed nucleotide sequence of 99% homology with the corresponding regions of rat cDNA sequence. The full length sponge cDNA was cloned in an bacterial expression vector (pET32a(+)], and the expressed protein of molecular weight ~25kDa was purified through chromatographic techniques. To characterize the biological activity of the 182kDa related sponge cDNA, it was cloned into a mammalian expression vector (pCDNA3.1 (-). Direct injection of the recombinant vector into the apex region of the rat heart resulted in substantial decrease of antioxidant enzymes such as SOD, GPX, GRD, XDH and Catalase with a subsequent increase in the levels of free radicals. Polyclonal antibodies raised against the purified protein in rabbits could nullify the decreased viability of human lymphocytes and HT29 colon cancer cell lines in cell cytotoxicity assay tested in the presence of the purified protein in cultures invitro. These data suggest that this particular 182kDa related sponge protein might play a crucial role in host defensive mechanism by enhancing free radical production.
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