Characterization,Expression Profile,and The Response Of OlApoA-Ⅰ Gene To Dietary Cholesterol And Immune Stimuli In Medaka(Oryzias Latipes)

Apolipoprotein A-Ⅰ(ApoA-Ⅰ)is essential for the atheroprotection and the metabolism of cholesterol in mammals.It also involves the immune functions of mammals,including humans and fishes.ApoA-Ⅰ exists on chromosome eleven(11)in humans and encodes a specific protein named Apolipoprotein A-Ⅰ.It was first isolated from human plasma HDL,followed by some non-human mammals and fishes.Medaka(Oryzias latipes)is a small fish suitable as a model for the study of ApoA-Ⅰ.In this study,the identification,characterization,and expression profile of medaka ApoA-Ⅰ(OlApoA-Ⅰ)was done in adult tissues and embryos of medaka.Moreover,the effect of dietary cholesterol on growth performance and OlAopA-Ⅰ expression was examined for the first time in medaka.In addition to these,the response of OlApoA-Ⅰ to immune stimuli,lipopolysaccharide(LPS),and polyinosinic-polycytidylic acid(Poly I:C)was also investigated.Isolation and amplification of OlApoA-Ⅰ ORF were performed by using the designed primers ApoA-Ⅰ-F(5’-ATGAAGGTGTTTGTGGTGCTA-3’)and ApoA-Ⅰ-R(5-CTCAGAGGATGGAGAAACAGT-3’)targeted on the predicted ApoA-Ⅰ sequences.Blast analysis and sequence alignment were done to identify the OlApoA-Ⅰ gene in medaka.Characterization of OlApoA-Ⅰ was made by using some bioinformatic tools.Reverse-Transcriptase Polymerase Chain Reaction(RT-PCR)and Quantitative Real-Time Polymerase Chain Reaction(qRT-PCR)were used to determine the expression profile of OlApoA-Ⅰ mRNA.Fishes were fed with a cholesterol-rich diet of different concentrations(Control with no free cholesterol,Experimental group 1;Exp.1 with a medium concentration of free cholesterol,and Experimental group 2;Exp.2 with a high concentration of free cholesterol)for one month.Changes in the weight and length of the young fishes in three groups were measured weekly during the experimental period.The expression of OlApoA-Ⅰ mRNA after feeding with experimental diets was measured with qRT-PCR.Lipopolysaccharide(LPS),polyinosinicpolycytidylic acid(Poly I:C),and Phosphate Buffer Saline(PBS)was intraperitoneally injected into 150 adult fishes divided into three groups,and tissues were collected at one to ten days post-injection(dpi)and subjected to qRT-PCR to determine the OlApoA-Ⅰ mRNA expression after immune challenges.99.60% identity among the experimental and NCBI GenBank sequences was evidenced in this study.The rooted phylogenetic analysis of the ApoA-Ⅰ protein sequence of fourteen different species showed five well-supported clades,each of which formed a monophyletic group with good posterior probabilities.The phylogenetic analysis also suggested a close relationship of OlApoA-Ⅰ with the Poecilia reticulata ApoA-Ⅰ and a distant relationship with Salmo salar,on the other hand.The result of the synteny analysis revealed that the gene ApoA-Ⅰ is well conserved from fish to mammals,including humans.OlApoA-Ⅰ possesses252 amino acids(aa)and consists of a short signal peptide of 17 aa at the Nterminal end,followed by a Pfam: apolipoprotein domain of 185 aa.Olapo A-Ⅰ is a secretory protein that exists outside of the cell membrane,as evidenced by the presence of a short signal peptide at the N terminal end.OlApoA-Ⅰ protein does not have any transmembrane helices.The Prot Scale analysis suggested that OlApoA-Ⅰ is a highly hydrophilic protein and involves different body functions.The secondary structure bears mostly alpha(α)helices and few coils but no beta(β)sheet.The tertiary structure consists of two antiparallelly arranged polypeptide chains,each of which bears several right-handed α-helices,a few coils,four O-glycosylation sites,and twenty binding sites.N-glycosylation sites and disulfide bridges are absent in OlApoA-Ⅰ.The expression of OlApoA-Ⅰ was recorded in all the adult tissues examined.However,the expression was highest in the intestine followed by the liver.Besides,the expression was also detected throughout the blastula stage until hatching during embryogenesis.Although a high-cholesterol diet did not impair the growth of young fish,it did cause considerable deposition of fat in the abdomen.At first,this diet reduced intestinal OlApoA-Ⅰ mRNA levels,but at the end of the research,those levels increased.On the other hand,OlApoA-Ⅰ mRNA was down-regulated in the liver,but initially elevated and later dropped in muscle and fat.Additionally,OlApoA-Ⅰ mRNA was up or down-regulated in immune organs like the liver,intestine,kidney,and spleen in adult fish after immunological activation with LPS and Poly I:C.This research concluded that OlApoA-Ⅰ is a secretory protein and exists outside the cell membrane.The expression of this gene in adult tissues is ubiquitous and is a zygotic gene in medaka embryos.The growth performance of young medaka does not affect by dietary cholesterol but causes excessive fat deposition in the abdomen resulting in obesity and capable of influencing the expression of OlApoA-Ⅰ in a tissue-specific manner.OlApoA-Ⅰ responds to immune stimuli.OlApoA-Ⅰ expression in response to dietary cholesterol and immune stimuli hints at a possible role in cholesterol metabolism and immune response.