Molecular bases of low production rates of apolipoprotein B-100 and truncated apoB-82 in a mutant HepG2 cell line generated by targeted modification of the apolipoprotein B gene

In subjects with familial hypobetalipoproteinemia heterozygous for truncate d forms of apolipoprotein B, both apoB-100 and the truncated forms are prod uced at lower than expected rates, We studied the mechanism of low levels o f apoB in a cell model produced by targeted modification of the apob gene o f HepG2 cells, One of the three alleles of apob was found to be targeted. T he targeted cells expressed apoB-100 and B-82. The media of mutant cells co ntained 56% of the levels of apoB-100 present in the media of wild-type (WT ) HepG2 cells. ApoB-82 was present at 11% of the apoB-100 levels in mutant cell media. An 85-kD protein (apoB-15) representing the N-terminal fragment of apoB was also secreted, but only in the mutant cell media. We examined the mechanism of low levels of apoB-82. Cellular apoB-82 mRNA was 11% of ap oB-100 mRNA, lower than the 33% expected, but consistent with relative leve ls of apoB-82 in the media. ApoB mRNA transcription in WT and the mutant ce lls did not differ, while the levels of apoB-82 mRNA in nuclei and polysome s were 46% and 12% of the levels of apoB-100 mRNA? respectively, suggesting that the lower levels of apoB-82 mRNA were due to altered message stabilit y. In a pulse/chase experiment with [S-35] methionine, at zero time of chas e, the amounts of apoB-100 in mutant cells was 66% that of WT levels, consi stent with the modification of one allele, The fractions of newly synthesiz ed apoB-100 secreted into the media at 2 h were 10% in the mutant cells and 19% in the WT cells, suggesting greater presecretory degradation of apoB-1 00 in the mutant cells. Thus, low levels of mutant apoB-82 mRNA gave rise t o the lo tv levels of apoB-82, while low levels of apoB-100 were due to low rates of secretion.