Microsomal Triglyceride Transfer Protein Binding and Lipid Transfer Activities Are Independent of Each Other, but Both Are Required for Secretion of Apolipoprotein B Lipoproteins from Liver Cells

Abstract

Recent studies indicate that microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) interact physically via two specific binding sites located within the amino-terminal globular region of apoB100. The first site is thought to be within the first 5.8% of the amino-terminal sequence, and the second site is between 9 and 16% of the amino-terminal sequence. It is not clear from prior studies whether these sites have unique or overlapping functions. Furthermore, there are no data differentiating between lipid transfer and potential chaperone functions of MTP. In the present study we have attempted to further characterize the physiologic interaction between apoB and MTP and to determine the relationship between the binding and lipid transfer aspects of the interaction. HepG2 cells were transiently transfected with apoB cDNAs, and MTP binding to apoB polypeptides was determined by two-step immunoprecipitation. MTP bound equally well to apoB polypeptides with (apoB13, 16,␤, apoB34, and apoB42) or without (apoB16, apoB13, and 16 or apoB13, 13, and 16) ␤ sheet domains. When proteasomal degradation of newly synthesized apoB polypeptides was blocked, MTP binding to all of the apoB polypeptides was only modestly affected by lipid availability and was independent of MTP-associated lipid transfer. Furthermore, MTP did not bind directly to a portion of the first ␤ sheet domain. We created two apoB constructs (apoB16 del and apoB34 del ) by deleting the first 210 amino acids of apoB16 and apoB34. These apoB polypeptides, therefore, lacked the putative first MTP binding site. MTP binding to apoB16 del and apoB34 del was decreased significantly. However, the secretion of apoB16 del was not different from apoB16, whereas the secretion of apoB34 del was impaired significantly. Our results indicate that the interaction between MTP and apoB involves independent binding and lipid transfer activities but that both activities are required for the secretion of apolipoprotein B from liver cells. Microsomal triglyceride transfer protein (MTP)1 is a heterodimer consisting of protein disulfide isomerase and the 97-kDa-large subunit and is found mainly in the endoplasmic reticulum (ER) lumen of liver and intestinal cells (1-3). The essential role of MTP in the translocation of apolipoprotein B (apoB) across the ER membrane and the assembly of apoB with lipids has been demonstrated clearly by co-expressing apoB and MTP in cells that do not normally express either protein and by the characterization of mice lacking MTP. The expression of apoB alone in cells without MTP resulted in the intracellular degradation of nascent apoB and no secretion; co-expression of apoB with MTP resulted in the secretion of apoB on lipoprotein particles (4 -6). Furthermore, the inhibition of MTP activity in cultured liver cells blocked the assembly and secretion of apoB-containing lipoproteins (7-11). We demonstrated that inhibition of MTP activity was associated with increased ubiquitination of apoB, particularly when proteasomal degradation was ...

Recent advances in physiological lipoprotein metabolism

“…MTP is a heterodimeric protein complex possessing lipid transfer activity, which functions in the small intestine and liver to transfer lipids to assist in the formation of primordial apoB lipoproteins. It has been shown to have three domains: 1) an apoB binding domain; 2) a lipid transfer domain; and 3) a membrane association domain [46]. The initial incorporation of lipids into apoB by MTP may prevent apoB from degradation.…”

Transmembrane Lipid Transfer Is Crucial for Providing Neutral Lipids during Very Low Density Lipoprotein Assembly in Endoplasmic Reticulum

“…It was clearly shown that newly synthesized apoB in cells lacking MTP failed to associate with lipids and subsequently the apoB was degraded (8). It is also reported that MTP has a molecular chaperon-like activity (9) and contributes to TG transfer from cytosol to lumen of the endoplasmic reticulum (10,11).…”

Molecular screening of the microsomal triglyceride transfer protein: association between polymorphisms and both abdominal obesity and plasma apolipoprotein B concentration

“…MTP catalyses the assembly of triglyceride-rich plasma lipoproteins (VLDL) and chylomicrons by carrying lipid from endoplasmic reticulum membrane to apoB particles in the lumen of endoplasmic reticulum (Olofsson et al 1999). The assembly of lipoproteins involves a physical binding of apoB and the larger subunit of MTP, but the precise mechanism remains to be elucidated (Bradbury et al 1999;Gretch et al 1996;Liang and Ginsberg 2001;Wetterau et al 1990).…”