A truncated species of apolipoprotein B, B-83, associated with hypobetalipoproteinemia.

Over the past five years, several laboratories have used a variety of transgenic and gene-targeted mice to study apoB. These studies have helped in 1 ) generating new mouse models suitable for investigating the genetic and environmental factors affecting atherogenesis; 2 ) providing systems for investigating apoB structure/function relationships; 3 ) understanding the regulation of apoB gene expression in the intestine; 4 ) delineating a critical role for apoB expression in mouse embryonic development; 5 ) yielding insights into the "physiologic rationale" for the existence of the two different forms of apoB, apoB-48 and apoB-100, in mammalian metabolism; and 6 ) providing basic insights into mechanisms involved in the human apoB deficiency syndrome, familial hypobetalipoproteinemia. -Kim, E., and S. G. Young. Genetically modified mice for the study of apolipoprotein B.

Section: A Gene-targeted Mouse Model For Understanding Mechanisms In the Human Apob Deficiency Syndrome Familial Hypobetalipoproteinemiamentioning

confidence: 53%

Genetically modified mice for the study of apolipoprotein B

Kim

Young

1998

“…The low relative levels of both apoB-82 and apoB-100 resembled plasma levels of truncated apoBs and apoB-100 in FHBL human and mouse heterozygotes. For truncations, these range from 5-25% of apoB-100 (38)(39)(40)(41)(42). For apoB-100, levels range from 30-50% of normal (3,4).…”

Section: Secretion Of Apobmentioning

confidence: 99%

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

Srivastava¹,

Srivastava²,

Averna

et al. 1999

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 [ 35 S] 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. K., N. Srivastava, M. Averna, A. B. Cefalu, and G. Schonfeld. 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.

“…Two lines of observations suggest that a minimum size be required for N-terminal fragments of apoB to efficiently participate in second-step assembly into full-sized VLDL. Studies of human subjects with familial hypobetalipoproteinemia (21)(22)(23)(24)(25), showed that truncated apoB corresponding to apoB-86, -50, -46, -40 and -39 all were found in the VLDL density range, albeit with increasing proportions of the shorter peptides in denser particles. In contrast, apoB-32.5, -31, and -27 were found only in the HDL density range, suggesting an inability to undergo secondstep assembly for these very short peptides.…”

mentioning

confidence: 99%

Rat McA-RH7777 cells efficiently assemble rat apolipoprotein B-48 or larger fragments into VLDL but not human apolipoprotein B of any size

Xiao

Elovson

Schumaker

2000

Studies of truncated apoB peptides in human subjects with familial hypobetalipoproteinemia, as well as of puromycin-generated spectra of nascent apoB peptides in rat and hamster liver, suggest that a minimum size is required for N-terminal fragments of apoB to be efficiently assembled into full-sized VLDL. We report here results of experiments undertaken to examine this phenomenon in greater detail by expressing individual carboxyl-truncated human apoB constructs in McArdle cells. Thus, apoB-29, -32, -37, -42, -47, -53, -70 and full length apoB-100 were transiently expressed in rat McA-RH7777 hepatoma cells, or human apoB-31 and apoB-53 were stably expressed in the same cells, and the secreted VLDL particles were characterized by kinetic gradient ultracentrifugal flotation. Calibration with rat plasma VLDL subfractions showed that about 90 and 50%, respectively, of lipoprotein particles containing endogenous rat B-100 and B-48 floated between fractions 2-8 of the 11-fraction gradient. This corresponds to the normal VLDL diameter range of about 47 to 28 nm, with the remaining half of rat B-48 recovered as HDL particles in the 1.1 g/ml range. In contrast, regardless of their size, only 2-5% of any of the truncated human apoB peptides expressed in these cells was recovered in the VLDL region of the gradient. The remaining 95 ؉ % of the lipoproteins were found as high density particles; as previously found in other systems the densities of the latter were inversely related to their peptide chain-length. Furthermore, transiently expressed full-length human apoB-100 was inefficiently secreted as VLDL by these cells, with the remainder appearing as LDL-sized particles. Thus, although we showed that McA-RH7777 cells secreted endogenous rat apoB as normal-sized VLDL, we found them unsuitable for our original purpose of using human apoB fragments to further define effects of apoB size on VLDL assembly. These cells appeared unable to efficiently use any size of human apoB for that process. Pulse-labeled untransfected McA-RH7777 cells chased in the presence of puromycin did, however, show a sharp decline in VLDL assembly efficiency for endogenous nascent rat apoB peptides shorter than B-48, similar to that originally found in normal rat liver. -Xiao, Q., J. Elovson, and V. N. Schumaker. Rat McA-RH7777 cells efficiently assemble rat apolipoprotein B-48 or larger fragments into VLDL but not human apolipoprotein B of any size.