| α-tocopherol transfer protein (α-TTP) is a protein that exhibits a marked ligand specificity and selectively recognizes of α-tocopherol and plays a key role in regulating concentrations of vitamin E. Despite the central roles that α-TTP plays in maintaining vitamin E adequacy, the available information is insufficient for a complete understanding of the mechanisms that how α-TTP is regulated by vitamin E supplementation. The studies of α-TTP have been mainly focused on human, mice and rats, however, little is known about ovine α-TTP. In the present study, α-TTP was first investigated in sheep and especially focused on the regulation of ovine α-TTP by dietary vitamin E supplementation and its possible mechanisms.Experiment1:Molecular cloning and analysis of the full-length cDNA of ovine α-TTP gene. The full-length cDNA of ovine p a-TTP gene was cloned from the liver tissue of Aohan fine-wool sheep by using of rapid amplification of complementary DNA ends. Bioinformatic analysis was then applied to analyze the gene and protein sequences of ovine α-TTP. As a result, the ovine α-TTP gene was1098bp in nucleotide which contained23bp5’-untranslated region (UTR),226bp3’-UTR and849bp open reading frame (ORF) that encoded a basic protein of282amino acids. Further bioinformatic analysis indicated that the ovine α-TTP was calculated to have a molecular mass of32.01kDa and it had a Secl4p-like lipid-binding domain. Besides, the ovine α-TTP was not a transmembrane protein. According to its gene and protein sequences, the ovine α-TTP had a high homologous of both nucleotide and amino acid sequences with the corresponding α-TTP gene from other mammals, however, the homologies were relatively low between sheep and other non-mammals.Experiment2:Effect of dietary vitamin E supplementation on the expression levels of ovine α-TTP. Thirty-five local male lambs of Tan sheep,20to30days after weaning, were randomly divided into five groups (n=7). The sheep were fed with diets supplemented with0(E0),20(E20),100(E100),200(E200) and2000(E2000) IU sheep-1d-1vitamin E for120days. The result showed that sheep fed with a high vitamin E diet (E2000) had significantly (P<0.05) higher plasma vitamin E concentration. Similarly, E200and E2000groups had significantly (P<0.05) higher hepatic vitamin E and α-TTP protein level compared to the control group (E0), however, no significant change was observed in ovine α-TTP gene expression among groups. Overall, the result indicated that high levels of vitamin E supplementation significantly (P<0.05) increased the concentrations of vitamin E in the plasma and liver as well as ovine liver α-TTP protein level, however, dietary vitamin E supplementation did not affect ovine α-TTP gene expression.Experiment3:Preparation of monoclonal antibody for ovine α-TTP. The amino acid sequenc of ovine α-TTP was first analyzed and two peptide immunogens with high specificity were selected and artificially synthesized. Then sheep α-TTP monoclonal antibody was produced by using lymphocyte hybridoma monoclonal antibody technology. In the end, the produced monoclonal antibody was purified and confirmed by different assays. Besides, immunohistochemical studies were also applied to analyze the distribution of a-TTP in ovine liver and the ovine a-TTP expression levels under different vitamin E supplementation. The result showed that a specific monoclonal antibody was successfully obtained which was proved to be effective in recognizing ovine a-TTP. Immunochemical staining also showed that a-TTP staining was mainly cytosolic and the result of immunohistochemistry revealed that no significant effect of vitamin E on ovine a-TTP distribution and expression levels in liver cells were observed.Experiment4:In order to study the mechanism of how ovine a-TTP gene expression was regulated by vitamin E, microarray technology was applied to screen differential expression genes in ovine liver among groups with different dietary vitamin E supplementation. On the other hand, the a-TTP vector was expressed in HepG2cell and a-TTP related genes were also screened by using microarray technology. Based on this, the function and pathway of both vitamin E and a-TTP related genes were analyzed in a comprehensive way to clarify how ovine a-TTP gene expression was regulated by vitamin E. Results suggested that547gene probes which might interact with vitamin E in ovine liver were obtained. Besides,323gene probes were identified to be regulated after a-TTP was expressed in HepG2cell. Based on the analysis of these genes, it was speculated that vitamin E might affect a-TTP gene expression by modulating the oxidation level or affecting genes implicated in protein kinase C signaling, or regulating the expression of transcription factors. Besides, vitamin E might also regulate a-TTP gene expression by regulating genes that related to the uptake and degradation of tocopherols, or genes which connected to adhesion, inflammation and apoptosis as well as some translocation-related genes.Experiment5:In order to elucidate how ovine a-TTP protein expression was regulated by vitamin E, ovine a-TTP interacting protein was screened using the method of co-immunoprecipitation coupled with mass spectrometry.10proteins that might interact with ovine a-TTP were identificated and these proteins were mainly involved in transportation, energy metabolism, gene expression regulation and stress reaction. However, whether they interact with ovine a-TTP in vivo or not still needs to be verified. It was possible that vitamin E might regulate a-TTP protein expression by acting as a ligand or by modulating the oxidative stress level. It was also very likely that vitamin E could affect a-TTP protein expression via changing the conformation of some translocation-related factors.Collectively, the present study investigated the effect of dietary vitamin E supplementation on ovine a-TTP expression for the first time and analyzed its mechanism in a comprehensive way. The findings will greatly enhance our understanding of the biological functions of ovine a-TTP and regulation mechanism of vitamin E transportation. |
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