Microsomal triglyceride transfer protein activity is not required for the initiation of apolipoprotein B-containing lipoprotein assembly in McA-RH7777 cells

We previously demonstrated that the N-terminal 1000 amino acid residues of human apolipoprotein (apo) B (designated apoB: 1000) are competent to fold into a three-sided lipovitellin-like lipid binding cavity to form the apoB lipid pocket without a structural requirement for microsomal triglyceride transfer protein (MTP). Our results established that this primordial apoB-containing particle is phospholipid-rich (Manchekar, M., Richardson, P. E., Forte, T. M., Datta, G., Segrest, J. P., and Dashti, N. (2004) J. Biol. Chem. 279, 39757-39766). In this study we have investigated the putative functional role of MTP in the initial lipidation of apoB: 1000 in stable transformants of McA-RH7777 cells. Inhibition of MTP lipid transfer activity by 0.1 mu M BMS-197636 and 5, 10, and 20 mu M of BMS-200150 had no detectable effect on the synthesis, lipidation, and secretion of apoB: 1000-containing particles. Under identical experimental conditions, the synthesis, lipidation, and secretion of endogenous apoB100-containing particles in HepG2 and parental untransfected McARH7777 cells were inhibited by 86-94%. BMS-200150 at 40 mu M nearly abolished the secretion of endogenous apoB100-containing particles in HepG2 and parental McA-RH cells but caused only 15-20% inhibition in the secretion of apoB: 1000-containing particles. This modest decrease was attributable to the nonspecific effect of a high concentration of this compound on hepatic protein synthesis, as reflected in a similar (20-25%) reduction in albumin secretion. Suppression of MTP gene expression in stable transformants of McARH7777 cells by micro-interfering RNA led to 60-70% decrease in MTP mRNA and protein levels, but it had no detectable effect on the secretion of apoB: 1000. Our results provide a compelling argument that the initial addition of phospholipids to apoB: 1000 and initiation of apoB-containing lipoprotein assembly occur independently of MTP lipid transfer activity.