| The liver is present in all vertebrates, and is typically the largest solid organ in the body. It plays a number of vital functions, including processing of nutrients from ingested food, plasma protein synthesis, hormone production, and detoxification. Emerging evidences also suggest that the liver is an immunological organ playing an important role in innate immunity, especially in acute phase response (APR). APR is a group of physiological processes occurring soon after the onset of infection, trauma, inflammatory processes, and some malignant conditions. Although genes specific to or enriched in the liver have been well investigated, those involved in APR have been poorly studied. On the other hand, expression of a large number of liver-specific or enriched genes has been shown to be regulated by the liver-specific transcription factors, hepatocyte nuclear factor 1 (HNF-1), HNF-3, HNF-4, HNF-6, and CAAT/enhancer binding protein (C/EBP). In particular, studies have reported the regulation of APR gene expression by HNF-4, C/EBP, and STATs. However, if genes involved in APR are also controlled by the liver-specific transcription factors, such as HNF-4 and C/EBP, have been less systematically explored. A better understanding of the regulatory network of APR gene expression will contribute to developing safe and effective means to manipulate this very important clinical response in mammals. Due to its evolutionarily unique position transient from invertebrates to vertebrates, amphioxus has been regarded as a model animal for understanding the origin of vertebrate; and its hepatic cecum, the pouch that protrudes forward as an outpocketing of the digestive tube and extends along the right side of the posterior part of the pharynx, has long been considered to be the homologous organ of vertebrate liver. However, if amphioxus shares vertebrate liver-specific genes, remains largely elusive. If so, do they display an expression pattern and regulatory network similar to those observed in vertebrates? Moreover, does their expression in response to challenge with lipopolysaccharide (LPS) bear resemblance to that in vertebrates? Studies as such will definitely shed more light on the scenarios of evolutionary origin of liver and APR. The primary aims of this study were to identify the liver-specific genes in amphioxus, and to examine their tissue (hepatic cecum)-specific expression patterns. The responsiveness of hepatic cecum-specific (in amphioxus) and liver-specific (in zebrafish) gene expressions to LPS challenge is also compared to uncover the similarities, if any, between APRs in these two animals. Additionally, by searching the promoter region of each hepatic cecum-specific gene, the putative binding sites of some well-known liver-specific transcription factors are also identified to delineate the regulatory network in the "pre-hepatic" APR in amphioxus with only hepatic cecum. By combining global genome survey and qRT-PCR data sets, here, we clearly demonstrate the presence of 58 vertebrate (zebrafish) liver-enriched genes in amphioxus (hepatic caecum-enriched genes) that are expressed in a tissue-specific manner in the hepatic caecum, the homolog of liver. Among these 58 hepatic caecum-enriched genes,52 genes respond to lipopolysaccharide challenge, which show similar expression profiles in both zebrafish and amphioxus. In addition, searching for binding sites for HNF and APR-associated transcription factors in promoter sequences for all the 58 hepatic caecum-enriched genes and the 52 APR-related genes suggests that both HNF factors and APR-associated transcription factors in amphioxus form regulatory networks similar to those observed in zebrafish, regulating the hepatic caecum-enriched genes and APR-related genes, respectively, via binding to their binding sites in the promoter regions. These similarities in liver/hepatic caecum-enriched genes, APR and regulatory networks between amphioxus and zebrafish supports that the hepatic caecum in amphioxus is the "pre-hepatic" organ homologous to vertebrate liver, and acts as an immunological organ, playing an important role in APR.Protein 4.1 (also called band 4.1) was originally identified as an abundant protein of human erythrocyte, and was regarded as an essential component for the maintaining of cell shape and integrity. It provides the connections between the skeleton and the plasma membrane in the erythrocytes, but also plays an important role in several cell functions in many other tissues/organs, including the bone marrow. cerebellum, lungs, testes and thymus. So far, little information is available regarding protein 4.1 in the lower animals. The cDNA encoding an amphioxus protein 4.1 (BbP41) was cloned from Qingdao amphioxus Branchiostoma japonicum. It contained a 1590 bp open reading frame corresponding to a deduced protein of 529 amino acids with a predicted molecular mass of approximately 59.3 kDa. The deduced BbP41 protein has 47.0%-60.8% identity with its homologues from a variety of organisms. The BbP41 protein also has the conserved domains of FERM, FA and CTD, but it lacks the SABD domain. Phylogenetic analysis showed that the vertebrate 4.1R,4.1G, 4.1N and 4.1B proteins are each grouped together, with BbP41 protein falling at the base of vertebrate protein 4.1 clade, suggesting that the divergence of vertebrate and invertebrate protein 4.1 gene probably occured prior to the split of invertebrate/vertebrate from a common ancestor around 550 million years ago, and the BbP41 protein might be the archetype of the vertebrate band 4.1 protein. The genomic DNA sequence of BbP41 contained thirteen exons and twelve introns, while the genomic structure of vertebrate protein 4.1 genes showed much more complexity. qRT-PCR showed that BbP41 transcript was abundant in the notochord, gill, hind-gut, ovary and testis. The His-BbP41 recombinant protein has been successfully expressed in Escherichia coli and purified. Western blot analysis showed that the His-BbP41 protein has a native molecular mass of approximately 66 kDa, and BbP41 has a high homology with human 4.1R protein. ELISA and His-Pull Down analysis showed that the recombinant His-BbP41 protein also can bind to both actin and spectrin, demonstrating the similar function as vertebrate protein 4.1.
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