Apolipoprotein B-containing lipoprotein assembly in microsomal triglyceride transfer protein-deficient McA-RH7777 cells

Abstract

Apolipoprotein (apo) B has a fundamental role in the transport and metabolism of plasma triacylglycerols (TAGs) and cholesterol and is synthesized primarily in hepatocytes and enterocytes ( 1-3 ). ApoB is present as a single molecule per lipoprotein particle ( 4 ) and exists in two forms, apoB100 (the full-length protein) and apoB48 (the N-terminal 48% of apoB100). The two forms derive from the same gene by a posttranscriptional modifi cation of the apoB mRNA at codon 2,153 that converts a glutamine codon to a stop codon ( 5 ). Studies by Xie et al. ( 6 ) provided the fi rst biologically plausible role for a mammalian intestinal apoB mRNA editing mechanism. ApoB100 is an essential structural component for the formation and secretion of VLDL and is essentially the only apoprotein component of LDL ( 3,7,8 ). ApoB48 is required for the formation and secretion of chylomicrons and is expressed in mammalian intestine and in the liver of some nonhuman mammals ( 3, 9 ).The processes involved in the assembly of apoB-containing lipoproteins in the liver are complex and are regulated at multiple levels throughout the secretory pathway. The addition of lipids to apoB is widely thought to occur in two steps ( 3, 10, 11 ). The fi rst step involves the addition of small amounts of lipids to apoB as it is translated and translocated into the lumen of the endoplasmic reticulum, preventing its degradation and allowing for the formation of a partially lipidated small preVLDL particle in the HDL density range ( 3,11,12 ). In the second step, this preVLDL particle acquires the bulk of its core lipids and is converted

Phospholipid Transfer Protein Plays a Major Role in the Initiation of Apolipoprotein B-containing Lipoprotein Assembly in Mouse Primary Hepatocytes

“…We demonstrated that BMSl97636 and BMS-200150, potent inhibitors of MTP activity (32), had no effect on the synthesis and secretion of apoB:1000-containing particles (11). In a subsequent study (12), we used MTP-deficient McA-RH7777 cells and demonstrated that the near complete absence of MTP had no effect on the synthesis and secretion of apoB:1000 (12). Likewise, identical levels of labeled lipids were found associated with apoB:1000-containing particles secreted by parental and MTP-deficient McA-RH7777 cells (12).…”

“…Although MTP has a distinct preference for TAG and cholesteryl esters, it also has a weak PL transfer activity (30), which has been suggested to be sufficient for the initiation of apoB particle assembly in nonhepatic COS7 cells (31). To test this potential role of MTP in cells of hepatic origin, we undertook comprehensive studies that included the use of MTP inhibitors (11) and rat hepatoma McA-RH7777 cells with 98% deficiency in MTP at both the mRNA and protein levels (12). We demonstrated that BMSl97636 and BMS-200150, potent inhibitors of MTP activity (32), had no effect on the synthesis and secretion of apoB:1000-containing particles (11).…”

“…transfer of lipids to apoB:1000, of MTP (6). In subsequent comprehensive studies in rat hepatoma McA-RH7777 cells, we used MTP inhibitors (11) as well as microRNA-mediated MTP-deficient McA-RH7777 cells (12) and demonstrated that the initial addition of PL to apoB:1000 is independent of MTP lipid transfer activity. Based on these results, we hypothesized that phospholipid transfer protein (PLTP) is a plausible mediator of this very early step in apoB-containing particle assembly.…”

“…Mouse 35 S-labeled endogenous apoB100 and apoB48 were immunoprecipitated using polyclonal antibodies to rat apoB100 as described (11,12). The 35 S-labeled proteins were extracted from Protein G and resolved by 4 -12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiogra-phy (20).…”

Selective Hepatic Insulin Resistance, VLDL Overproduction, and Hypertriglyceridemia

“…Extrusion of the first 1000 residues (βα1 domain) and association with phospholipid within the endoplasmic reticulum (ER) lumen are necessary for the initiation of particle assembly and may not require the activity of MTP. 7 The length of the domain of B100, necessary for MTP-dependent particle assembly, is still under debate. 8,9 Multiple interactions of apoB with MTP and lipid transfer form partially lipidated particles through size-dependent linear lipidation and result in formation of low-density lipoprotein (LDL)-VLDL2 particles that act as precursors to VLDL1.…”