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 truncated forms of apolipoprotein B, both apoB-100 and the truncated forms are produced at lower than expected rates. We studied the mechanism of low levels of apoB in a cell model produced by targeted modification of the apob gene of HepG2 cells. One of the three alleles of apob was found to be targeted. The targeted cells expressed apoB-100 and B-82. The media of mutant cells contained 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 apoB-100 mRNA, lower than the 33% expected, but consistent with relative levels of apoB-82 in the media. ApoB mRNA transcription in WT and the mutant cells did not differ, while the levels of apoB-82 mRNA in nuclei and polysomes 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 stability. In a pulse/chase experiment with [35S] methionine, at zero time of chase, the amounts of apoB-100 in mutant cells was 66% that of WT levels, consistent with the modification of one allele. The fractions of newly synthesized 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-100 in the mutant cells. Thus, low levels of mutant apoB-82 mRNA gave rise to the low levels of apoB-82, while low levels of apoB-100 were due to low rates of secretion.
Srivastava
R A
RA
Division of Atherosclerosis, Nutrition and Lipid Research, Department of Internal Medicine, Washington University, Saint Louis, MO 63110, USA.
Srivastava
N
N
Averna
M
M
Cefalu
A B
AB
Schonfeld
G
G
D000483:Alleles; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D018390:Gene Targeting; D006801:Humans; D006995:Hypobetalipoproteinemias / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D017931:DNA Primers; D012333:RNA, Messenger