Abstract
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.
Highlights
The processes involved in the assembly of Apolipoprotein B (apoB)-containing lipoproteins in the liver are complex and are regulated at multiple levels throughout the secretory pathway
Because microsomal triglyceride transfer protein (MTP) both binds to apoB and transfers lipids [33], this study was a logical continuation of our previous work and focused on the following important question: “is MTP involved in the initial addition of PL to nascent apoB:1000 and initiation of apoB-containing lipoprotein particle assembly?” To address this question, we tested the effects of two well characterized inhibitors of MTP lipid transfer activity, BMS-197636 and BMS-200150 [13, 34], on the de novo synthesis, lipidation, and secretion of apoB:1000 in stable transformants of McARH7777 cells
We tested the effects of two known inhibitors of MTP lipid transfer activity, BMS-197636 and BMS-200150 [13, 17, 51], on the synthesis, lipidation, and secretion of apoB:1000-containing particles stably expressed in McA-RH cells
Summary
Materials—Horse serum (HS) and antibiotic-antimycotic were obtained from Invitrogen. Tris/glycine gels were obtained from Invitrogen. At the start of experiments, maintenance media were removed; cells were washed twice with PBS and were incubated for 17 h in serum-free DMEM (for McA-RH cells) or MEM (for HepG2 cells) containing either [3H]glycerol (7 Ci/ml of medium) or [14C]oleic acid (0.4 mM) bound to 0.75% BSA. The incorporation of [3H]glycerol or [14C]oleic acid into various lipid moieties of apoB-containing lipoproteins secreted into the medium and accumulated in the cells was determined by immunoprecipitation or by nondenaturing gradient gel electrophoresis (NDGGE) as described below. Immunoprecipitation—The 35S-labeled proteins secreted into the conditioned media and accumulated in the cells were immunoprecipitated using monospecific polyclonal antibody to human or rat apoB100 coupled to protein G-Sepharose CL-4B as described previously [27, 39]. MTP mRNA level was determined by RT-PCR, and MTP protein level was assessed by immunoblotting [41] using polyclonal antibody to bovine MTP 97-kDa large subunit
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