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

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