Characterization, Evolution And Functional Analysis Of One Of The Scavenger Receptor In Amphioxus Branchiostoma Japonicum

Scavenger receptor class B (SRB) are transmembrane glycoproteins and contain three members:CD36, SR-BⅠ and SR-BⅡ. SRB can bind a wide variety of ligands and express on many cells and tissues, so they can mediate diverse normal physiological and pathological processes, such as innate immune defence, internalization of apoptotic cells, atherothrombotic diseases and other cardiovascular diseases. The structures, functions, sequences and expressions of many kinds of SR-B have been studied, yet little is known to date about it in the amphioxus Branchiostoma japonicum. Amphioxus or lancelet, a cephalochordate, located between the invertebrates and vertebrates, and is becoming an emerging model organism for insights into the origin and evolution of vertebrates. In this paper, we report cloning, evolution, expression and functional analysis of BjCD36gene in amphioxus Branchiostoma japonicum. The main results are as follows:1Five SR-B homologues (SRB-a, b, c, d, e) were identified in amphioxus Branchiostoma floridae, and they encoded proteins with length of480,445,912,229and159amino acids, respectively, but only theree of them (SRB-a, b, c) contained the CD36domain through the analysis of Conserved Domain Database in NCBI. The alignment of multiple protein sequences among SR-B revealed that the SRB-a was more similar to SR-B than other four genes and the similarity between SRB-a and CD36is more high than SRB-a and SR-BⅠ/Ⅱ. Also, analysis of exon-intron organization showed that the exon-intron organization of SRB-a was very similar to the that of vertebrates (especially zebrafish). So the SRB-a gene was indentified as the homology of CD36. Moreover, analysis of the exon-intron organization, the intron type and the chromosome locus revealed that CD36gene was conserved during evolution. 2The cDNA encoding SRB-a was obtained by RACE-PCR in amphioxus B. japonicum, and was named BjCD36in this paper. The full-length cDNA of BjCD36was1711bp long, containing an open reading frame (ORF) of1482bp which encoded a protein of494amino acids. BjCD36showed90%of sequence identity with SRB-a from amphioxus B.floridae in amino acid level.3Real-time PCR and in situ hybridization demonstrated that BjCD36transcript was abundant in hepatic caecum, and at a slightly lower level in other tissue. Whole mount in situ hybridization showed that BjCD36transcript was widely expressed in the early embryos and the1-day larvae, but was expressed in the front one third of the full-length body in2-day larvae, where the gill and hepatic caecum will form later during development.4Real-time PCR indicated that challenge with E. coli (0h-48h) resulted in an up-regulation of BjCD36expression in the gut-free bodys of amphioxus, and10μg/ml or20μg/ml of LTA-induced BjCD36up-regulation in the FG cells transfected with BjCD36was found. These results suggested that BjCD36may be involved in the host immune defense. In order to detect the response of BjCD36F-a to feeding, the adult amphioxus was fed with algae S. platensis, then was challenged with hungry. It was found that BjCD36was up-regulated after feeding, then a down-regulation of BjCD36expression in the gills was decteced with hungry challenge, indicating the roles of BjCD36in the feeding control. Together these studies identify and characterize a conserved gene that is important in the fundamental process of immune and feeding regulation.