Studies on the assembly and secretion of apolipoprotein B containing lipoproteins

Abstract

Lipoproteins are water-soluble particles of lipids and proteins, which deliver water-insoluble lipids from intestine or liver to target tissues where the lipids are used as fuel or storage. Lipoproteins consist of a core of hydrophobic lipids surrounded by an amphipatic monolayer of polar lipids and specific proteins, apolipoproteins. There exist several lipoprotein species. The triglyceride rich lipoproteins that are secreted by the liver are referred to as very low density lipoproteins (VLDL), while the triglyceride rich lipoproteins assembled in the intestine are called chylomicrons. Apolipoprotein B (apoB) is absolutely necessary for the assembly of VLDL and chylomicrons. ApoB exists in two forms: apoB-100 and apoB-48. ApoB-48 consists of the N-terminal 48% of apoB-100; and.it is coded for by the same gene as apoB-100. An increased secretion of VLDL is an important risk factor for developing atherosclerosis. Such increased production of VLDL occurs in for example type 2 diabetes and insulin resistance. This thesis deals with the assembly and secretion of lipoproteins containing apoB. The assembly of VLDL occurs in two major steps. The first step is the cotranslational lipidation of apoB resulting in a partly lipidated apoB, which in the second step is converted to VLDL by the addition of the major amount of lipids. I have been able to separate these two steps with Brefeldin A. Brefeldin A treatment results in the assembly of partly lipidated apoB, of which the apoB-100 is loosely associated with the microsomal membrane. Neither apoB-100 nor apoB-48 VLDL is assembled, thus Brefeldin A inhibits the second step of the VLDL assembly. The Brefeldin A effect is reversible, the partly lipidated apoB can than assemble VLDL upon removal of Brefeldin A, i.e. the membrane-associated form of apoB-100 is the precursor of apoB-100 VLDL. It is well established that MTP (the microsomal triglyceride transfer protein) is required for the production of VLDL. Absence of MTP leads to a total loss of apoB containing lipoproteins in the plasma (a-betalipoproteinemia). We have shown that MTP is needed during the formation of the apoB-100 VLDL-precursor, i.e. during the cotranslational lipidation of apoB-100 and during a short time posttranslationally. MTP is however not needed during the second step, i.e. when the pre-VLDL aquires the major amount of lipids. The availability of fatty acids and the biosynthesis of triglycerides are of importance for the second step in the VLDL-assembly. Therefore the importance of L-FABP (the liver fatty acid binding protein), a protein thought to modulate the fatty acid uptake and utilisation, was investigated. Inducing L-FABP-expression caused a decrease in b-oxidation of fatty acids, suggesting that L-FABP sequester fatty acid to some other process. No effect on lipid biosynthesis or lipoprotein assembly could be seen though. We also did a first attempt to identify sequences of apoB that are needed for the VLDL-assembly. Recent studies have suggested that the C-terminal part of apoB-48 is of importance for this process. This part consists of amphipatic a-helices. To answer the question if the general lipid affinity of these a-helices is of importance for the VLDL-assembly we made mutations calculated to decrease the predicted lipidaffinity in two of the most hydrophobic a-helices. The mutation in the helix consisting of residue 2127-2152 in apoB was associated with a significant decrease in the expression of the protein and a subsequent decrease in the VLDL secretion. This was not seen for the mutation in the 2046-2078 sequence. Both the mutations should lead to a similar decrease of the calculated hydrophobicity of the region there in. Thus the effect of the mutation in 2127-2152 is more specific than just general hydrophobicity.