Study On The Genes And Their Functions Of The Complement-like Components From Scallops

Complement system, as the important part of innate immunity and one of the major effecter arms of immune responses, plays a critical role in innate immune defense of invertebrates. The study on invertebrate complement-like system is still on the beginning and the information about complement-like molecules in mollusks remains deficient. In the present study, Chlamys farreri thioester-containing protein (CfTEP), Clq-domain-containing protein (CfC1qDC), C-type lectins (Cflec-3, Cflec-4 and Cflec-5) and Argopecten irradians C-type lectin (AiCTL-6) and fibrinogen-related protein (AiFREP) were studied by molecular biotechnologies including RACE, RT-PCR and recombinant protein expression. The full-length cDNA sequences of CfTEP, CfC1qDC, Cflec-3, Cflec-4, Cflec-5, AiCTL-6 and AiFREP were of 4616,777,2256,2080,1412,1063 and 990 bp, respectively. The open reading frame (ORF) encoded a polypeptide consisted of a signal peptide, with anα2-macroglobulin domain in CfTEP, a Clq domain in CfClqDC, three or four carbohydrate recognition domain (CRD) in Cflec-3, Cflec-4, one CRD in Cflec-5 or AiCTL-6, and a fibrinogen-related domain in AiFREP. In healthy scallops, CfTEP and Cflec-4 mRNA was only expressed in hepatopancreas and gonad, while others could be found in all the tissues detected. All of their mRNA expression levels could be remarkably up-regulated by Listonella anguillarum challenge. The genomic DNA of CfTEP consisted of 40 exons and 39 introns, and seven different transcripts were produced by alternative splicing. These transcripts displayed different expression pattern in gonads in response to different bacterial challenges. The recombinant CfClqDC protein displayed a significantly strong activity to bind LPS. The recombinant Cflec-3 agglutinated Pseudomonas stustzeri in the calcium ions-dependent D-mannose-binding manner. The recombinant Cflec-5 agglutinated Pichia pastoris GS115 in the calcium ions-independent D-mannose-binding manner. The recombinant AiCTL-6 agglutinated bacteria Escherichia coli TOPI OF', Micrococcus luteus and Staphylococcus aureus. And the recombinant AiFREP agglutinated not only bacteria L. anguillarum, E.coli JM109 and M. luteus, but also chicken and human erythrocytes. These activities were calcium ions-dependent and could be inhibited by acetyl group-containing carbohydrates.These results collectively suggested that these molecules were involved in the immune response against microbe infections and functioned as pattern recognition receptors in the innate immune system of scallops. They shared structural and functional similarity with the mammalian complement components, including C3, Clq, MBL and ficolin, and they might be the ancestor forms of these molecules contributing to nonself-recognition in invertebrates. It would provide clues for elucidating the evolution origin of the complement components and contribute to further study on the primary and simple complement-like system in scallops. Meanwhile, it would be helpful for understanding the scallops'immune defense mechanisms and provide new information for the enrichment and development of marine invertebrate immunology.