Analysis Of Phylogenesis And Expression Patterns Of C1q, C3 And G10 In Amphioxus And Zebrafish

Complement system is composed of soluble proteins in the serum and tissue fluid,membrane-associated proteins and complement receptors.According to their function,complement system can be divided into three types.The first type is the intrinsic component in body fluid playing a role in complement activation of enzymatic chain reaction,including eight complement components(C1-C9),D factor, B factor and P factor.The second type consists of the regulatory molecules during the complement activation,including the soluble protein in the tissue fluid such as C1 esterase inhibitor,C4bp,I factor,S protein and membrane-associated protein in the cell membrane such as membrane cofactor,decay accelerating factor,homologous restriction factor.The third type contains the receptors on cell surface to mediate the active fragments of the complements or the receptors to regulate the protein with biological effect,these receptors include Clq receptor,C3b/C3b receptor,H factor receptor,C3a,C5a receptor.The complement system in the tissue fluid usually remains in the inactive state,and performs its biological activity only when actived by an activator.The complement system plays an important role in the innate immune system as well as in the acquired immune system.The complement system has four main functions:bacteriolysis and cytolysis,neutralizing and dissolving virus, regulating immune adherence and mediating inflammation.C3 is the most abundant complement protein in the immune serum,which consists ofα- andβ-chains connected by a single disulfide bond.As a multifunctional protein,C3 can interact with a number of complement factors and non-complement factors.C3 is the central component of complement system which is the cross point of the three activation pathway,and mediates the cascade amplification effect.C1q(Complement component 1,q subcomponent,C1q) is the recognition protein in the classical pathway of complement system.Human C1q consists of 18 polypeptides,including chain A,B,C.C1q can extensively interacts with self- and nonself-ligands through the trimmer gC1q domain,which leads to the activation of the classical pathway.C1q plays a role in immune system as lectin-like component in invertebrate and lower vertebrate.The phylogenetic analysis and genomic structure analysis of C3 has been performed,and the expression patterns of C3 gene in embryonic development and adult tissues have been performed as well.The C3 protein alignment indicated that AmphiC3 protein share low identity(about 25%-30%) with homologs in other species. However,all of them have the "RRRR" domain and the C3a anaphylatoxin domain which characterized with "CC--C--C--CC".The relatively higher homology with the mammals indicated that the variability of C3 becomes restricted when it evolved into mammal.The result of the phylogenetic analysis indicated C3 of amphioxus is in the transit site between of invertebrate and vertebrate,which was consistent with the position of amphioxus in animal classification.The genetic structure analysis shows that the C3 of human,mouse and zebrafish is composed of forty-one exons and 40 introns,and every exon has the same size.AmphiC3 is composed of 40 exons and 39 introns.The 22nd exon of the amphioxus is corresponding to the combination of the 22nd and 23rd exons of zebrafish.The combination of the 23rd and 24th exon of the amphioxus is corresponding to the 24th exon of the zebrafish gene.The 39th exon is corresponding to the combination of the 39th and 40th exons.The size of other exons are mostly similar to those of human,mouse and zebrafish.In situ hybrization was performed in adult amphioxus and we found that the expression of the AmphiC3 appeared in neurons that around central canal epitheliums of pharynx,macrophage in the coelom,ciliated columnar epithelial cells of branchial plate,gut,ovary,testis,epidermic cells at the metapleural fold and macrophage in the lymph cavitys of the metapleural fold.We also found some slender signal of C3 in endostyle.The conserved sequence and the expression pattern of AmphiC3 indicated that AmphiC3 may be involved in the immune response of amphioxus and play a similar role.During the development of amphioxus embryo,the expression of amphiC1q-like is detected in every stage.Its expression was observed in presumptive notochordal mesoderm,paraxial mesoderm,developing neural plate and differentiating gut. Continually,its expression was found in the developing and newly formed notochord, somite as well as in theintestinal wall.In the larval phase,the expression mainly located in the gut,gill,and differentiating tailbud.When it comes to the adult tissues, the expression pattern of Clq-like was similar to the C3,but in endostyle the expression of C1q-like was much stronger.Comparative study indicated the similar expression pattern of C1qc gene and C3 gene in the adult tissues of amphioxus and zebrafish,and the expression signal was mainly detected in ovary,retina,brain,liver and gut.So it suggested that the function of C1q and C3 in amphioxus and zebrafish may be similar.G10 protein was originally discovered as a maternal protein in Xenopus oocyte. G10 protein of Mus musculus has 93%identity with the G10 protein of Xenopus. Timothy H1a discovered G10 homologous protein in human umbilical vein endothelial cells,with 93%identity with Xenopus oocyte.The G10 gene was expressed ubiquitously in cell lines of epithelial cells and lobus intermedius.In addition,the G10 polypeptide sequence was highly conserved in evolution and was also expressed in inferior organisms such as yeast and C.elegans.The phylogenetic analysis,genomic structure analysis and expression pattern of G10 gene in embryo development and adult tissues have been performed,the results of which suggested that G10 of amphioxus is in the transit site between of invertebrate and vertebrate,which was consistent with the position of amphioxus in animal classification.Results from whole-mount in situ hybridization showed that the expression of amphiG10 was detected in differentiating ectoderm and mesoderm. With the development proceeding,the expression signal was mainly found in epidermis,somite,gut,newly formed neural tubes and notochord.Then,its expression was observed in epithelial cells of gut and gill.The expression pattern of amphiGlO in adult tissue was similar to that of C1q-like and C3.The expression pattern of zebraG10 was strikingly similar to that of C1qc and C3.And the only dissimilarity was the absence of the expression signal of G10 gene in retina.G10 gene is likely to be involved in immune activity.