Apolipoprotein B sequence requirements for hepatic very low density lipoprotein assembly. Evidence that hydrophobic...

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-beta), 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 N-acetyl-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.