Identification, Expression And Regulation Of Branchiostoma Japonicum Ceruloplasmin Gene With An Emphasis On Origin Of Liver

Ceruloplasmin (Cp), a copper-containing a-glycoprotein is synthesized mainly in liver. Apo-Cp is first synthesized followed by the binding of copper ion along with the incorporation of a polysaccharide moiety to form holo-Cp and released into blood. Cp is widely distributed in invertebrates and vertebrates, an important metalloproteinase for animal and human life. Notably, among all its functions, the following two are the most important:(1) plasma copper carrying, where 95% of copper in the circulation system is bound to Cp; and (2) controlling iron homeostasis by functioning as ferroxidase, and more importantly controlling the free radical production.Cp is an indispensible member of the multi-copper oxidases (MCOs). MCOs is a category of enzymes that are able to oxidize their substrate to two water molecules by concomitant reduction of dioxygen. To date, three multi-copper oxidases have been identified in vertebrates:Cp, hephaestin and zyklopen. Due to The family has ferrous oxidase activity, it can effectively oxidize Fe2+ to Fe3+, they all have a high specific affinity with iron. Cp is wildly distributed in tissue to ensure a proper iron turnover process in the system to prevent toxic effects caused by Fe2+, which, through the Feton and Haber-Weiss reactions, contribute the generation of free radicals. In this way, Cp act as a key component in the homeostatic regulation of blood iron levels.The cephalochordate amphioxus has recently been repositioned at the base of the chordate phylum, which occupies a nodal position from invertebrates to vertebrates. Amphioxus has become an emerging model organism for insights into the origin and evolution of vertebrates. Many studies suggested the presence of liver-like (hepatic caecum) tissues in Amphioxus and Cp is mainly synthesized in the liver. Copper-bound Cp is released to the blood by the liver. liver plays a major role in iron storage and detoxification in vertebrate. However, if a similar regulatory function does the hepatic caecum have remains an open question, and is worth exploring. Cp plays a key role in cellular iron metabolism including iron storage and detoxification, which has been identified in awide range of organisms including plants and animals. However, little information is available regarding Cp in the protochordates to date. Despite extensive study on Cp in vertebrates, information regarding expression and regulation of Cp genes is rather limited in invertebrates.Here we report identification of Cp gene in Branchiostoma japonicum Branchiostoma belcheri. Cp encoded a deduced protein of 1178 amino acids with the conserved motif for cu-oxidase center typical of vertebrate Cp. Sequence comparison revealed that the predicted protein was 42.7% to 46.1% identical with Cp isolated from the vertebrates and 20.7% to 49.7% identical with Cp from the invertebrates. Comparison of the 3D structures of Branchiostoma japonicum and Homo sapiens Cp revealed a striking similarity although they both shared only 45.8% identity. These suggest that Branchiostoma japonicum Cp may play a similar role as Homo sapien Cp do. The Real-time PCR revealed that it was ubiquitously expressed in B. belcheri. and it was abundantly expressed in the hepatic caecum in a tissue-specific manner. In addition, Cp expression was up-regulated respectively, following exposure to LPS at both transcriptional and translational levels. Similarly, exposure to iron resulted in increase in Cp in the humoral fluids. These suggest that Cp seems a protein with a dual function functioning in both immune response and iron metabolism.In conclusion, this study highlights that Branchiostoma japonicum Cp is structurally and functionally similar to Cp in the vertebrates. All these provide functional evidences, supporting the notion that the hepatic caecum is the liver homologue, playing a key role in maintaining iron homeostasis in Branchiostoma japonicum.