Identification, Expression And Functional Characterization Of Complement Components C1q-like And C3a Molecules In Amphioxus

Amphioxus, a basal chordate, has long been regarded a model organism for studying the origin and evolution of vertebrates. The study on the complement system of amphioxus will certainly contribute to elucidating the origin and evolution of vertebrate complement. The complement system, which helps Abs and phagocytic cells to clear pathogens from an organism, is a central component of innate immunity and a link between innate and adaptive immunity. There are three pathways by which the complement system can be activated:the classical pathway, the lectin pathway, and the alternative pathway. We report here the cloning, expressing and functional analysis of the complement genes C1q and C3a.After analyzing the C1q-like genes in amphioxus genome, we selected one orthologue of vertebrate Clq for further study. We cloned this gene from Branchiostoma japonicum and named it BjC1q. BjC1q contains the key residues of human C1q binding to IgG and C1r-C1s. It is predominantly expressed in the hepatic caecum, hindgut, and notochord, and is significantly up regulated following challenge with bacteria or LTA and LPS, suggesting that BjC1q is associated with immune response in amphioxus. In this study, recombinant expression, ELISA, western blot, hemolytic assay, and affinity chromatography were used to test whether recombinant BjClq could initiate the classical pathway. BjC1q can assemble to form the high molecular weight oligomers necessary for binding to proteases C1r/C1s and for complement activation, and binds human C1r/C1s/mannan-binding lectin-associated serine protease-2 involved in the cleavage of C4/C2, and C3 activation. Moreover, BjClq and its globular head domain specifically interact with LTA and LPS, but BjClq displays little lectin activity. Importantly, BjClq binds with human IgG, resulting in the activation of the classical complement pathway. This is the first report showing that a C1q-like protein in invertebrates is able to initiate classical pathway.More importantly, the function of BjC1q in vivo was needed to be studied, and more experiments were performed to further test whether amphioxus complement system could be activated by BjClq. At first, we identified the BjClq-interaction protein through pull-down assay and peptide mass fingerprinting analysis, which showed that the interaction protein contained three Ig domain and was named as BjIgSF. The interaction of BjIgSF and BjC1q was similar with that of IgG and C1q, which is able to activate classical pathway. Additionally, the results of C4 and C3 deposition assay showed that B. japonicum humoral fluids (HF) was able to cleave C4 and C3, while heat-inactivated HF (which inactivates serine proteases) or C1q-like proteins depleted HF (which lacks C1q-like proteins) was not. C1q-like proteins depleted HF supplemented with BjC1q was able to cleave C4 and C3, suggesting the presence of an interaction of BjC1q with serine proteases in HF. Together, these data implicate that BjC1q can associate with proteases in HF and form BjClq-protease complex, triggering the cleavage of human C4 and C3. We propose that amphioxus possesses a C1q-mediated complement system. It also suggests a new scenario for the emergence of the classical complement pathway, in contrast to the proposal that the lectin pathway evolved into the classical pathway.We demonstrated that C3a from the invertebrate chordate Branchiostoma japonicum, BjC3a, was similar to vertebrate C3a possessing potential antibacterial activity, as revealed by sequence analysis and computational modeling. The antibacterial activity of BjC3a was definitely confirmed by antibacterial assay. Additionally, recombinant BjC3a, like vertebrate C3a, was capable of inducing sea bass macrophage migration and enhancing macrophage phagocytosis and respiratory burst response. Most of the biological effects mediated by the anaphylatoxin C3a are suppressed by removal of its C-terminal arginine through cleavage by serum protease carboxylpeptidase N, generating the inactive C3a-desArg peptide. However, C3a-desArg still retains some of the immunological functions of C3a. We also demonstrated that recombinant BjC3a-desArg (generated by removal of the C-terminal arginine), like mammalian C3a-desArg, retained the immunological activities of BjC3a such as antibacterial and respiratory burst-stimulating activities, indicating that the immunological functions of C3a-desArg were conserved throughout chordate evolution. Altogether, our findings show that invertebrate (amphioxus) BjC3a is able to interact with vertebrate (sea bass) macrophages and mediate immune activities, suggesting the emergence of the inflammatory pathway of the complement system similar to that of vertebrates in the basal chordate amphioxus.