Apolipoprotein B Sequence Requirements for Hepatic Very Low Density Lipoprotein Assembly

Abstract

We studied the structural requirements of apolipoprotein (apo) B for assembly of very low density lipoproteins (VLDL) using rat hepatoma McA-RH7777 cells expressing human apoB (h-apoB). Recombinant h-apoB48, like endogenous rat apoB48 (r-apoB48), was secreted as VLDL in addition to high density lipoproteins (HDL) by transfected cells, indicating that the N-terminal 48% of apoB contains sequences sufficient for VLDL assembly. Truncation of the C terminus of h-apo-B48 to -B42 or -B37 had little effect on the ability of apoB to assemble VLDL, whereas truncation to -B34 or -B29 markedly diminished or abolished VLDL formation. None of the truncations affected the integration of apoB into HDL. To determine whether the ability to assemble VLDL is governed by apoB length or by sequences beyond apoB29, we created chimeric proteins that contained human apoA-I and a segment derived from between the C-terminal 29 and 34%, 34 and 37%, or 37 and 42% of apoB100. The resulting chimeras, namely AI/B29 -34, AI/ B34 -37, and AI/B37-42, were secreted by the transfected cells as lipoproteins with buoyant density (d < 1.006 g/ml), electrophoretic mobility (pre-␤), and size characteristics of human plasma VLDL. The chimeras could assemble discrete VLDL particles devoid of endogenous r-apoB100, and could actively recruit triglycerides and phospholipids into the lipoproteins. However, these chimeras were secreted inefficiently. Pulse-chase analysis showed that less than 5% of the newly synthesized AI/B proteins were secreted, and more than 70% was degraded intracellularly. Degradation of the chimeras could be blocked by the cysteine protease inhibitor Nacetyl-leucyl-leucyl-norleucinal, but the treatment did not enhance their secretion. Protease protection analysis of microsomes isolated from transfected cells indicated that >65% of AI/B chimeras (compared with <25% of r-apoB100) were inaccessible to exogenous trypsin. These data suggest that the recruitment of large quantities of triglycerides during VLDL formation is not governed simply by apoB length, but is mediated by short hydrophobic sequences ranging from 152 to 237 amino acids (3-5%) of apoB. The existence of multiple such hydrophobic sequences within apoB48 may facilitate efficient assembly of hepatic VLDL particles.

The Activity of Microsomal Triglyceride Transfer Protein Is Essential for Accumulation of Triglyceride within Microsomes in McA-RH7777 Cells

“…3). This is because the previously commonly used density ultracentrifugation technique, although satisfactory for revealing the two-step process for B48-VLDL assembly (4,7,33), does not allow detection of size changes in VLDL particles containing large apoB (i.e. B80 or B100).…”

“…The cells were homogenized using a ball-bearing homogenizer (20 passages) (33), and the postnuclear supernatant was obtained by centrifugation (10,000 ϫ g, 4°C, 10 min). Heavy microsomes were isolated from the postnuclear supernatant by centrifugation using a microcentrifuge (16,000 ϫ g, 4°C, 15 min), and subsequently the light microsomes and cytosol were separated by centrifugation using a Beckman TLA-100.4 rotor (400,000 ϫ g, 4°C, 16 min).…”

Missense Mutations in APOB within the βα1 Domain of Human APOB-100 Result in Impaired Secretion of ApoB and ApoB-containing Lipoproteins in Familial Hypobetalipoproteinemia

“…Both Leu 343 and Arg 463 residues are located within the predicted ␣-helical domain of apoB, which contains sequence elements shown to be important for proper folding of apoB, for the physical interaction between MTP and apoB, and for lipoprotein assembly. Transfection studies have suggested that apoB segments containing sequences lacking the ␤␣1 domain were unable to be secreted (13) or else secreted poorly (14). Mutational analysis has identified critical disulfide linkages within the ␤␣1 domain that are essential for efficient secretion of apoB and apoB-containing lipoproteins (15,16,18).…”

“…Transfection studies show that apoB segments lacking the ␤␣1 domain are either unable to be secreted (13) or poorly secreted (14). Mutagenesis experiments show that correct disulfide bond formation within the ␤␣1 domain is required for efficient secretion of apoB (15)(16)(17), and this requirement is independent of the lipidation state of apoB (18).…”

Proteolytic Degradation and Impaired Secretion of an Apolipoprotein A-I Mutant Associated with Dominantly Inherited Hypoalphalipoproteinemia

“…The labeling medium was then removed, and growth medium (DMEM plus 10% FBS) was added. Cells and medium were collected at the various time points up to 4 h after the initial pulse and harvested as described by McLeod et al (23). ApoA-I was immunoprecipitated from the media and cell lysates with anti-human apoA-I antisera (Roche Molecular Biochemicals) and protein G-Sepharose (Amersham Pharmacia Biotech) and then subjected to 12% SDS-PAGE.…”