Purification and characterization of microsomal triglyceride and cholesteryl ester transfer protein from bovine liver microsomes

Microsomal triglyceride transfer protein (MTP) transfers lipids to apolipoprotein B (apoB) within the endoplasmic reticulum, a process that involves direct interactions between apoB and the large subunit of MTP. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. We hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of halfnormal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the MTP:apoB ratio would be even lower). To test this hypothesis, we examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to our expectations, half-normal levels of MTP reduced the plasma apoB100 levels to the same extent (ϳ25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum rather than the MTP:apoB ratio is the critical determinant of lipoprotein secretion. Finally, we found that heterozygosity for an apoB knockout mutation lowered plasma apoB100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice. Microsomal triglyceride transfer protein (MTP)1 is expressed at high levels in the absorptive enterocytes of the intestine and hepatocytes, at moderate levels in the visceral endoderm of the yolk sac during embryonic development, and at relatively low levels in kidney and cardiac muscle (1-4). In each of these tissues, MTP plays a key role in the assembly and secretion of apoB-containing lipoproteins (1, 3, 5). MTP is located within the lumen of the endoplasmic reticulum (ER) and is thought to transfer lipids to apoB as that molecule is translated, allowing apoB to assume a proper conformation for forming a spherical lipoprotein with a neutral lipid core (6). In the absence of MTP, triglyceride-rich, apoB-containing lipoproteins cannot be assembled or secreted (3, 6, 7). The mechanism of MTP action has attracted considerable scrutiny. Of note, several lines of evidence have suggested that the assembly of lipoproteins involves a direct interaction between MTP and apoB. First, Wu et al. (8) demonstrated that apoB can be immunoprecipitated from the microsomes of cult...

Section: Methodsmentioning

confidence: 99%

Section: Microsomal Triglyceride Transfer Protein (Mtp)mentioning

confidence: 99%

A Deficiency of Microsomal Triglyceride Transfer Protein Reduces Apolipoprotein B Secretion

Leung¹,

Véniant²,

Kim³

et al. 2000

“…The activity was determined by evaluating the transfer of radiolabeled triacylglycerol between two populations of unilamellar vesicles as described previously (45,46). The donor and receiver vesicles were prepared by adding the appropriate amount of lipids to 500 l of chloroform followed by drying under a stream of nitrogen, rehydration, and probe sonication in 1. hydroxytoluene.…”

Section: Microsomal Triglyceride Transfer Protein Enzymatic Activity mentioning

confidence: 99%

Modification in Oxidative Stress, Inflammation, and Lipoprotein Assembly in Response to Hepatocyte Nuclear Factor 4α Knockdown in Intestinal Epithelial Cells

Marcil

Seidman

Sinnett

et al. 2010

“…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)(2)(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.…”

Section: Microsomal Triglyceride Transfer Protein (Mtp)mentioning

confidence: 99%

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

Liang

Ginsberg

2001

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 ...