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.