| Complement is an important component of the innate immune system. The mechanism of the complement of the vertebrates, especially in mammals, is clear. Research of complement in invertebrates, however, is only restricted to the Cephalochordata (amphioxus), Urochordata (ascidian), Echinodermata (sea sea urchin) or so. Amphioxus is the closest relative to vertebrate, and so it is an ideal model animal to research the development and evolution of the complement system. Our previous studies have shown that the complement system of the amphioxus Branchiostoma japonicum (formerly known as B. belcheri tsingtauense) is related with the lysis of erythrocytes and microbes via the activation of alternative pathway. However, if the complement system of amphioxus has the opsonic activity to enhance phagocytosis by phagocytes remains to be determined.As amphioxus lacks free circulating blood cells, we therefore used L. japonicus macrophages to test the opsonic activity of humoral fluid (HF) in this study. The opsonic ability was evaluated by the phagocytic ability of L. japonicus macrophages.Firstly, the yeast cells that had been incubated with humoral fluid (HF) of amphioxus were mixed with the macrophages isolated from sea bass and incubated, and then the phagocytosis was assessed. It was found that amphioxus HF was capable of markedly promoting the phagocytosis by sea bass macrophages. This indicated there is interplay between amphioxus HF with fish macrophage, i.e. amphioxus HF has an opsonic activity. The opsonic activity of amphioxus HF was in a dose-dependent manner. Significant enhancement of phagocytosis was still detectable even when the HF was diluted 4-folds.Secondly, the phagocytosis by sea bass macrophages was significantly reduced when the yeast cells were treated with amphioxus HF heated at 45℃for 30 min, a condition known to inactivate complement activity. This suggests a role of complement in the opsonic activity of HF. In agreement, the pre-incubation of HF with the antibody against Bf, a key enzyme involved in the AP activation, also led to a remarkable decrease in the opsonic activity of HF. Moreover, Western blotting revealed that there are C3 fragments monitored in the elution derived from yeast cells incubated with amphioxus HF. All these indicate that the amphioxus complement system is an important element involved in the opsonic activity, which can promote the sea bass macrophage phagocytosis by tagging yeast cells with C3 fragments via activation of alternative complement pathway.In conclusion, this study highlights the interplay between complement system of the lower animals (a deuterostome invertebrate) with macrophages of the vertebrates (bony fish). This also bolsters the notion that the complement system in both invertebrates and vertebrates shares a common ancestry.Lysozyme is an important component of innate immunity. It is classified into three types g-type, c-type and i-type lysozyme in animals group. Interestingly, amphioxus is the only species containing all the three types of the enzyme (all other animals have only one or two types of the enzyme). Therefore, it is worth studying if all the three lysozymes in amphioxus are functional. As the first step, we underwent the cloning and identification of the lysozyme genes from the amphioxus Branchiostoma japonicum.A cDNA of g-type lysozyme was isolated from B. japonicum, named AmpLyz-g, which was 1014 bp with an ORF of 801 bp. It encodes a polypeptide of 188 amino acid residues. The SignalP program detected a signal peptide of 15 amino acid residues. It contains a catalytic bacterial soluble lytic transglycosylase (SLT) domain, which was well conserved. Three amino acid residues that are considered as potentially important for the lysozyme catalytic activity (Glul53, Asp187 andAsp178) were found to be completely conserved in AmpLyz-g. The sequence of AmpLyz-g showed above 50% identity to that of fish. Moreover, Analysis of g type lysozyme genes show that AmpLyz-g contain 7 extrons. the first three extrons were detected no domain, while the other four extrons were consisted with the structure of vertebrates.We also cloned two full length cDNA of i type Lysozyme from Branchiostoma japonicum, named AmpLyz-i, which share the same ORF. The longer sequence is 1053 bp, while the shorter cDNA is 849 bp. Its ORF encodes 167 amino acid residues, and contain a conserved destabilase domain with three possible catalytic activity (Glu36, Asp49 and Asn120). AmpLyz-i show high identity to that of echinodermates, approximately 60%.Phylogenetic analysis of lysozymes showed that AmpLyz-g and AmpLyz-i were clearly grouped in the same clade as other g-type and i-type lysozymes respectively. The c-and i-type lysozymes clustered to one cluster, while the g-type lysozyme was probably the ancester of lysozyme family. |
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