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 microm BMS-197636 and 5, 10, and 20 microm 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 microm 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.
Dashti
Nassrin
N
Department of Medicine, Basic Sciences Section, Atherosclerosis Research Unit, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294; Department of Cell Biology, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294. Electronic address: ndashti@uab.edu.
Manchekar
Medha
M
Department of Medicine, Basic Sciences Section, Atherosclerosis Research Unit, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294.
Liu
Yanwen
Y
Department of Medicine, Basic Sciences Section, Atherosclerosis Research Unit, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294.
Sun
Zhihuan
Z
Department of Medicine, Basic Sciences Section, Atherosclerosis Research Unit, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294.
Segrest
Jere P
JP
Department of Medicine, Basic Sciences Section, Atherosclerosis Research Unit, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294; Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294.
D000418:Albumins / Q000378:metabolism; D000818:Animals; D053299:Apolipoprotein B-100 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004527:Egg Proteins / Q000235:genetics; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D054833:Isoindoles; D008099:Liver / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D010880:Piperidines / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D051381:Rats
D000418:Albumins; D053299:Apolipoprotein B-100; C101970:BMS 200150; D002352:Carrier Proteins; D004527:Egg Proteins; D007211:Indoles; D054833:Isoindoles; D010743:Phospholipids; D010880:Piperidines; D034741:RNA, Small Interfering; C106092:microsomal triglyceride transfer protein; C001549:lipovitellin