Role of Phospholipid Transfer Activity of MTP in Promoting Lipoprotein Assembly; Molecular Mechanisms and Structural Implications

Abstract

ApoB, MTP and lipids are essential for apoB-lipoprotein assembly and secretion. Deficiency in any of these three factors affects lipoprotein production. The main function of apoB-lipoproteins is to deliver fat to other tissues for normal physiologic activities; but their excess accumulation in the plasma induces atherosclerosis and an array of metabolic complications such as Type 2 diabetes and obesity. It is evident that approaches to reduce apoB-lipoprotein production would help treat dyslipidemias. Although the exact mechanism of apoB-lipoprotein assembly and secretion is unknown, it is believed that microsomal triglyceride transfer protein (MTP) transfers neutral lipids onto nascent apoB while it is co-translationally translocated into the endoplasmic reticulum (ER) to form primordial lipoproteins. The goal of this study was to obtain structure and function of MTP to elucidate the role of different lipid transfer activities of MTP in the biosynthesis of apoB-lipoproteins. We used two independent approaches. In the first approach, we used evolutionarily diverse MTP orthologs that differ in their lipid transfer properties. Second, we used missense mutations reported in human that display different disease phenotype to elucidate the importance of different amino acids, α-helices and β-sheets in the MTP molecule that are crucial for different lipid transfer properties.

Our evolutionary studies indicate that MTP evolved as a phospholipid transfer protein and acquired triglyceride transfer activity during a transition from invertebrate to vertebrate. Comparative in vitro studies revealed that Drosophila MTP (dMTP) could transfer phospholipids but does not transfer triglyceride. In contrast, human MTP (hMTP) transfers both phospholipids and triglycerides. The importance and exact role of these lipid transfer activities in apoB-lipoprotein assembly is not known. We set out to find the physiological gains endowed on MTP as it acquires triglyceride transfer activities. To delineate in vivo roles of these transfer activities, we expressed these orthologs in liver-specific MTP deficient mice that have low plasma and high hepatic lipids. MTP orthologs reduced hepatic lipids and partially restored plasma lipids in these mice. dMTP mice produced apoB100/apoB48 VLDL and phospholipid-rich apoB48 HDL lipoproteins. Lower plasma lipids in dMTP mice, compared to hMTP were due to increased degradation of apoB. hMTP increased association of triglycerides with apoB in the endoplasmic reticulum membranes, inhibited proteasomal degradation, and formed triglyceride-rich VLDL. These studies demonstrate that MTP’s phospholipid transfer activity prevents hepatosteatosis and maintains low plasma lipids by synthesizing triglyceride-poor VLDL and phospholipid-rich HDL particle. We posit that triglyceride transfer activity of MTP is dispensable and its specific inhibition could be used to lower plasma lipids.

Our missense mutation studies show that complete loss of phospholipid and triglyceride transfer activities abolish apoB secretion. This is consistent with the absence of apoB-lipoproteins in abetalipoproteinemia. Analysis of missense mutations found in hypobetalipoproteinemia showed that these mutants lacked phospholipid transfer activity and exhibited partial triglyceride transfer activity. These mutants supported secretion of apoB-lipoproteins but to a lower extent compared to normal MTP. Thus partial loss of triglyceride transfer activity and selective loss of phospholipid transfer activity in MTP could support apoB-lipoprotein assembly and secretion but to lesser extent thereby resulting in a less severe phenotype in individuals resembling heterozygous hypobetalipoproteinemia which is primarily caused by mutations in apoB rather than abetalipoproteinemia. Therefore, hypobetalipoproteinemia phenotype could be due to inefficient assembly and secretion of apoB-lipoproteins. It is likely that genetic screening for mutations in MTP besides apoB could potentially explain hypobetalipoproteinemia in subjects that do not carry mutations in apoB.

In short, our evolutionary and mutation studies suggest that both phospholipid and triglyceride transfer activities of MTP are required for optimum apoB-lipoprotein assembly. MTP has been a therapeutic target to lower hyperlipidemia; however available drugs globally inhibit MTP leading to adverse events. Recognition of different roles for phospholipid and triglyceride transfer activities suggested that these activities could be separated and targeted for differential inhibition. Domain specific antagonists rather than global inhibition of MTP could help identify better therapeutic approaches to combat hyperlipidemia, obesity and atherosclerosis.