Demonstration of a Physical Interaction between Microsomal Triglyceride Transfer Protein and Apolipoprotein B during the Assembly of ApoB-containing Lipoproteins

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: Discussioncontrasting

confidence: 56%

Section: Mtp Activity and Protein Levels In Liver Extracts From Diffementioning

confidence: 99%

A Deficiency of Microsomal Triglyceride Transfer Protein Reduces Apolipoprotein B Secretion

Leung¹,

Véniant²,

Kim³

et al. 2000

“…Despite the fact that MTP is characterized in great detail, relatively little is known about the molecular details of the role of this protein in the assembly of the lipoprotein. An interaction with apoB has been demonstrated (23,24) and suggested to involve the N-terminal globular domain of apoB, which is needed for the action of MTP (25,26). In a recent study, we demonstrated that MTP is necessary for the early events in the assembly of endogenous apoB-48 VLDL in the McA-RH7777 cells, but does not appear to be essential for the step in which the major amount of lipids was added to the particle (14).…”

Section: Discussionmentioning

confidence: 99%

The Microsomal Triglyceride Transfer Protein Catalyzes the Post-translational Assembly of Apolipoprotein B-100 Very Low Density Lipoprotein in McA-RH7777 Cells

Rustaeus

Stillemark

Lindberg

et al. 1998

118

In cells in which the lipoprotein assembly process had been inactivated by brefeldin A (BFA), membrane-associated apoB-100 disappeared without forming lipoproteins or being secreted, indicating that it was degraded. Reactivation of the assembly process by chasing the cells in the absence of BFA, gave rise to a quantitative recovery of the membrane-associated apoB-100 in the very low density lipoprotein (VLDL) fraction in the medium. These results indicate that the membrane-associated apoB-100 can be converted to VLDL.A new method was developed by which the major amount (88%) of microsomal apoB-100 but not integral membrane proteins could be extracted. The major effect of this method was to increase the recovery of apoB-100 that banded in the LDL and HDL density regions, suggesting that the membrane-associated form of apoB-100 is partially lipidated. We also investigated the role of the microsomal triglyceride transfer protein (MTP) in the assembly of apoB-100 VLDL using a photoactivatable MTP inhibitor (BMS-192951). This compound strongly inhibited the assembly and secretion of apoB-100 VLDL when present during the translation of the protein. To investigate the importance of MTP during the later stages in the assembly process, the cells were preincubated with BFA (to reversibly inhibit the assembly of apoB-100 VLDL) and pulse-labeled (؉BFA) and chased (؉BFA) for 30 min to obtain full-length apoB-100 associated with the microsomal membrane. Inhibition of MTP after the 30-min chase blocked assembly of VLDL. This indicates that MTP is important for the conversion of full-length apoB-100 into VLDL. Results from experiments in which a second chase (؊BFA) was introduced before the inactivation of MTP indicated that only early events in this conversion of full-length apoB-100 into VLDL were blocked by the MTP inhibitor. Together these results indicate that there is a MTP-dependent "window" in the VLDL assembly process that occurs after the completion of apoB-100 but before the major amount of lipids is added to the VLDL particle. Thus the assembly of apoB-100 VLDL from membrane-associated apoB-100 involves an early MTP-dependent phase and a late MTP-independent phase, during which the major amount of lipid is added.

“…Previous studies have demonstrated a physical interaction between MTP and the amino-terminal domain of apoB (20,21). It is possible therefore that discrete disulfidebonded loops within this domain form a critical recognition site for the initial interaction of MTP with the nascent apoB polypeptide chain.…”

Section: Cys Pairs 2 and 4 Are Essential For The Assembly And Secretimentioning

confidence: 99%

Identification of Cysteine Pairs within the Amino-terminal 5% of Apolipoprotein B Essential for Hepatic Lipoprotein Assembly and Secretion

Huang

Shelness

1997

There is growing evidence that the amino-terminal globular domain of apolipoprotein B (apoB) is essential for lipoprotein particle formation in the hepatic endoplasmic reticulum. To identify the structural requirements for its function in lipoprotein assembly, cysteine (Cys) pairs required to form the seven disulfide bonds within the amino-terminal 21% of apoB were replaced in groups or individually by serine. Substitution of Cys pairs required for formation of disulfide bonds 1-3 or 4 -7 (numbered from amino to carboxyl terminus) completely blocked the secretion of apoB28 in transfected HepG2 cells. To identify the specific disulfide bonds required for secretion, Cys pairs were mutated individually. Substitution of Cys pairs required for disulfide bonds 1, 3, 5, 6, or 7 had little or no impact on apoB28 secretion or buoyant density. In contrast, individual substitution of Cys pair 2 (amino acid residues 51 and 70) or 4 (218 and 234) severely inhibited apoB28 secretion and its capacity to undergo intracellular assembly with lipid. The same assembly and secretion defects were observed when these mutations were expressed as part of apoB50. These studies provide direct evidence that the ability of the internal lipophilic regions of apoB to engage in the recruitment and sequestration of lipid during translation is critically dependent upon a structural configuration contained within or affected by the amino-terminal 5% of the protein.Little is known about how the initial folding of apolipoprotein B (apoB) 1 influences its interactions with microsomal triglyceride transfer proteins (MTP) and other factors to initiate the process of hepatic apoB-containing lipoprotein assembly (1-3). Although incapable of assembling with appreciable lipid on its own (4 -7), the amino-terminal disulfide-bonded region of apoB may, nonetheless, be essential for lipoprotein assembly. Based on the use of dithiothreitol (DTT) to probe protein folding in vivo, the amino-terminal domain of apoB was shown to undergo a folding reaction that is completed before the translation and translocation of the internal lipophilic regions of apoB. If DTT was added to cells during the translation of this domain, thereby preventing its proper folding, apoB was incapable of engaging in lipoprotein assembly (8,9). Although these and other studies (7) are consistent with an essential role in the initiation of lipoprotein assembly, it is not known which, among the seven disulfide bonds present in the amino-terminal 21% of apoB (10), are required for this function. Furthermore, it could not be established whether the block in lipoprotein assembly caused by DTT was caused only by perturbed apoB aminoterminal folding or whether it also resulted from DTT-mediated inhibition of one or more assembly factors.In the current study, the relationship between the structure of the amino-terminal domain of apoB and its capacity to initiate lipoprotein assembly in vivo was examined by performing systematic mutagenesis to disrupt its normal pattern of disulfide bonding (10). I...