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J. Biol. Chem., Vol. 282, Issue 39, 28597-28608, September 28, 2007

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Microsomal Triglyceride Transfer Protein Activity Is Not Required for the Initiation of Apolipoprotein B-containing Lipoprotein Assembly in McA-RH7777 Cells^*

Nassrin Dashti¹, Medha Manchekar, Yanwen Liu, Zhihuan Sun, and Jere P. Segrest^¶

From the Department of Medicine, Basic Sciences Section, Atherosclerosis Research Unit, Department of Cell Biology, and ^¶Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294

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 µM BMS-197636 and 5, 10, and 20 µM 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 McA-RH7777 cells were inhibited by 86-94%. BMS-200150 at 40 µM 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 McA-RH7777 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.

Received for publication, January 9, 2007 , and in revised form, July 18, 2007.

^* This work was supported by the National Institutes of Health Grants HL084685 and PO1 HL34343. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Medicine, University of Alabama at Birmingham, 1808 7th Ave. South, BDB-D680, Birmingham, AL 35292-0012. Tel.: 205-975-2159; Fax: 205-975-8079; E-mail: ndashti{at}uab.edu.

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