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-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.
Highlights
We studied the structural requirements of apolipoprotein B for assembly of very low density lipoproteins (VLDL) using rat hepatoma McA-RH7777 cells expressing human apoB (h-apoB)
The current study has attempted to determine if the ability to assemble apoB48-VLDL is governed by length or by unique sequences of the protein, and provided evidence that VLDL assembly is probably mediated by hydrophobic, lipid-binding domains within apoB48
Results obtained from experiments with both the truncated h-apoBs and the AI/B chimeras clearly demonstrate that the full-length of apoB48 is not a prerequisite for VLDL assembly
Summary
Important roles in the assembly of triglyceride-rich LpB. Assembly of VLDL in the liver is a complex process perhaps occurring in multiple steps. Immunoreactive apoB has been detected on the cytosolic side of hepatic microsomal membranes (14 –17) It has been suggested by some [18], but disputed by others [19], that translocation pausing is essential for lipidation of the apoB polypeptides. While the relatively large truncated apoB forms (e.g. ՆapoB37) were invariably found in VLDL and low density lipoproteins (LDL) in addition to HDL, the short forms (e.g. apo-B31 and -B32.5) were found only in HDL and the d Ͼ 1.21 g/ml fractions [3] These data imply that apo-B31 and -B32.5 may not contain the sequences necessary for VLDL formation, whereas apoB37 does. We used five C-terminally truncated h-apoB forms and three chimeras containing h-apoB segments that were expressed in stably transfected McA-RH7777 cells to determine whether the sequence requirement for hepatic VLDL assembly is determined by apoB length or by domains within the protein. Our results suggest that the amphipathic  strands between the C termini of apo-B29 and -B42 (B29 – 42) play important roles in VLDL assembly, and segments (as short as 152 amino acids) derived from B29 – 42 are sufficient to mediate VLDL formation
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