The Evolution of Microsomal Triglyceride Transfer Protein and its Role During the Assembly of PaoB-lipoproteins.

Abstract

The microsomal triglyceride transfer protein (MTP) transfers lipids (triacylglycerols, cholesteryl esters, and phospholipids) in vitro and is required for the secretion of apoB-lipoproteins in vivo. Inhibitors of MTP lipid transfer activity significantly increase the degradation of apoB and reduce its secretion. Therefore, transfer of lipids to apoB by MTP is believed to be essential for the assembly of apoB-lipoproteins. In the following studies we explored the structures and activities of evolutionarily distinct MTP homologs in order to better understand how MTP might be assisting apoB-lipoprotein assembly. We describe specific and sensitive in vitro assays to measure MTP cholesteryl ester and phospholipid transfer activities using fluorescent lipids. These activities are dependent on MTP concentration and demonstrate saturation kinetics. In contrast to the human MTP that transfers all lipids, we observed that the Drosophila MTP is deficient in the transfer of triacylglycerols and cholesteryl esters but does transfer phospholipids. Similarly the zebrafish MTP, like the human MTP, transfers triacylglycerols while the C. elegans MTP lacks this activity. Despite differences in their abilities to transfer lipids, all MTP orthologs assisted in the assembly and secretion of human apoB48-lipoproteins. Furthermore, the amounts of apoB secreted with the assistance of different MTP proteins were augmented by increasing triacylglycerol synthesis (the addition of oleic acid or expression of acyl-CoA:monoacylglycerol acyltransferase and acyl-CoA:diacylglycerol acyltransferase proteins) and decreased by treating the cells with triacsin C, an inhibitor of triacylglycerol synthesis. We subsequently showed that the secondary and tertiary structures in the orthologs were highly conserved while their primary amino acid sequences were less conserved. Interestingly the bC domains as well as helices 4-6 in the a-helical domains demonstrated greater conservation in vertebrate MTPs than the other structural domains. Together these data reveal that the phospholipid transfer activity is the most ancient activity associated with MTP and is sufficient to generate an apoB-lipoprotein particle. Triacylglycerol transfer activity was acquired during evolution and is specific to vertebrate MTP proteins. This activity is associated with greater conservation in the bC domain and helices 4-6 in the a-helical domain. Therefore, we propose that the amino acid content contained in the bC domain as well as helices 4-6 might be critical for the robust triacylglycerol transfer activity in vertebrates.