Isolation and characterization of a LPS induced MD2-like protein in Ciona intestinalis

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Abstract

The MD2 (Myeloid Differentiation factor-2) protein belongs to the ML superfamily. This group of proteins contain a specific lipid binding domain (ML domain) that plays an important role in lipid recognition and metabolism. In vertebrates, MD-2 is involved in innate immune response as co-receptor in the LPS/TLR4 signaling pathway; MD2 recognizes and binds the bacterial lipid A and drives the TLR4 activation. Two TLR isoforms, CiTLR-1 and CiTLR-2, were identified in Ciona intestinalis with a TIR domain most similar to human TLR4 and TLR 6 respectively. Using a PCR-based subtractive hybridization strategy for isolation of differentially expressed genes between LPS-challenged and naïve C. intestinalis, we identified a full-length cDNA (855 bp) encoding for a 150 a.a. protein (CiMD-2-like). In silico analysis showed that the deduced protein contains a signal peptide (1-19 a.a.) and an E1/Der p2/Der f2/ML domain-MD2 related lipid recognition domain (21-148 a.a) with similarities to ML(MD-2 related Lipid-recognition) domain identified in MD-2 and NPC2 (Niemann-Pick disease type C2). Phylogenetic and structural analysis supported the close relationship with MD-2 and NPC2 suggesting that CiMD-2-like originated from a common ancestor gene. Furthermore, gene expression studies by Real-time PCR demonstrated that this cDNA is up-regulated after LPS injection in the body wall. In situ hybridization performed in controls and LPS-induced animals has shown that this gene is expressed in granular amebocytes, large granules hemocytes and URG (univacuolar refractile granulocyte) in pharynx, the main organ of the ascidian immune system
Original languageEnglish
Number of pages1
Publication statusPublished - 2015

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