Abstract
The emopamil binding protein (EBP) is an important enzyme participating in the final steps of cholesterol biosynthesis in mammals. A predictive gene EBP-like, which encodes the protein with a high identity to human EBP, was found in chicken genome. No regulatory mechanisms and biological functions of EBP-like have been characterized in chickens. In the present study, the coding sequence of EBP-like was cloned, the phylogenetic trees of EBP/EBP-like were constructed and the genomic synteny of EBP-like was analyzed. The regulatory mechanism of EBP-like were explored with in vivo and in vitro experiments. The biological functions of EBP-like in liver cholesterol biosynthetic were examined by using gain- or loss-of-function strategies. The results showed that chicken EBP-like gene was originated from a common ancestral with Japanese quail EBP gene, and was relatively conservative with EBP gene among different species. The EBP-like gene was highly expressed in liver, its expression level was significantly increased in peak-laying stage, and was upregulated by estrogen. Inhibition of the EBP-like mRNA expression could restrain the expressions of EBP-like downstream genes (SC5D, DHCR24, and DHCR7) in the cholesterol synthetic pathway, therefore downregulate the liver intracellular T-CHO level. In conclusion, as substitute of EBP gene in chickens, EBP-like plays a vital role in the process of chicken liver cholesterol synthesis. This research provides a basis for revealing the molecular regulatory mechanism of cholesterol synthesis in birds, contributes insights into the improvement of the growth and development, laying performance and egg quality in poultry.
Highlights
As an important lipid molecule, cholesterol plays essential roles in growth and differentiation of eukaryotic cells, as well as in regulating the properties of plasma membranes in cells by affecting membrane fluidity, phase behavior, thickness, and permeability (Crockett, 1998; Simons and Ikonen, 2000)
Sequence alignment showed that the emopamil binding protein (EBP)-like coding sequence were successfully cloned with 100% identity to the EBP-like coding sequence predicted on NCBI GenBank (Supplementary Figure 2A)
This study for the first time verified the EBP-like gene in chickens, and revealed its evolutionary relationship, expression pattern, regulatory mechanism and biological functions
Summary
As an important lipid molecule, cholesterol plays essential roles in growth and differentiation of eukaryotic cells, as well as in regulating the properties of plasma membranes in cells by affecting membrane fluidity, phase behavior, thickness, and permeability (Crockett, 1998; Simons and Ikonen, 2000). Cholesterol is a vital precursor for hormones, bile acids, and lipoproteins production. Cholesterol synthesis in hepatocytes can be divided into three main stages catalyzed by functional specific enzymes (Sakakura et al, 2001; Lorbek et al, 2013). Acetyl coenzyme A was catalyzed to produce HMG-CoA by sulfur lyase and other enzymes, catalyzed by HMGCR (3Hydroxy-3-methylglutaryl-CoA reductase) to form mevalonate (MVA), which will be converted into isoprene pyrophosphate (IPP) during enzymatic reactions such as phosphorylation and decarboxylation. The product of the previous step was catalyzed by cyclase and oxygenase into lanosterol, which was reacted in multiple steps to produce cholesterol (Acimovicand Rozman, 2013). The emopamil binding protein (EBP) plays a key role in the final stage by acting as a D8-D7 sterol isomerase which converts Cholest8(9)-en-3beta-ol (8,9 choletenol) into cholest-7-en-3betaol (lathosterol)
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