Cloning, Expression And Evolution Characterization Of Bf/C2 Gene In Amphioxus Branchiostoma Belcheri

Amphioxus, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. Amphioxus represents the simplest model of the vertebrate morphogenesis, development pattern and genome structure and gives us a good animal model to study the origin of vertebrate immune system and the evolution of immune-related genes. In the absence of the vertebrate adaptive immune system based on highly specific antibodies and antigen receptors, amphioxus must rely on the efficient innate immune system to alleviate the burden of antipathogen defense. Complement system is a major component of the immunity. Complement activation via three overlapping pathways: the classical complement pathway (CCP), the alternative pathway (ACP), and the lectin complement pathway (LCP). Factor B plays a crucial role as the proteolytic subunits of the C3 convertase in alternative pathways which are mainly synthesized as inactive precursors in the liver. There are some reports about factor B or its homologous genes in human being, pongo, rat, xenopus, zebrafish, lamprey, ascidian, sea urchin, horseshoecrab and sea anemoneand a complement system operating via the alternative pathway (APC) similar to that of the vertebrate has been demonstrated in the primitive chordate amphioxus. However, there is no factor B gene reported in amphioxus. In this paper we report the cloning, expression and functional analysis of Branchiostoma belcheri factor B-like gene (BbBf/C2). The full-length cDNA of BbBf/C2 is 2362 bp with an open reading frame (ORF) of 2259 bp. The ORF of BbBf/C2 encodes a polypeptide of 752 amino acids with a calculated molecular weight of about 82.6 KDa. BbBf/C2 encoded a mosaic protein with three complement control protein (CCP) domains, a von Wilebrand factor A (vWFA) domain and a serine protease (SP) domain. Peculiarly, BbBf/C2 had an epidermal growth factor-like domain (EGF_CA) located between CCP1 and CCP2, therefore BbBf/C2 had a modular structure of CCP-EGF_CA-CCP-CCP-vWFA-SP, making it a novel member of Bf/C2 family proteins. Real-time PCR assay revealed that lipopolysaccharide (LPS) challenge resulted in a quick and continuously significant up-regulation of BbBf/C2 expression in the hepatic caecum, while BbBf/C2 was only shortly expressed in the hind-gut following LPS challenge though the expression level was temporarily higher than that in the hepatic caecum. Similarly, immuno-dot blotting showed that challenge with LPS triggered a significant elevation of BbBf/C2 synthesis in the hepatic caecum and hind-gut, with a higher rise in the former tissue. These indicate that both hepatic caecum and hind-gut may be involved in the immune response induced by LPS, but the hepatic caecum, like the vertebrate liver, is the primary tissue synthesizing BbBf/C2 in response to LPS challenge, thereby playing a major role in acute phase response.