Abstract
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.
Highlights
Studies of truncated apolipoprotein B (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 very low density lipoproteins (VLDL)
McA-RH7777 cells have been used previously to study VLDL assembly, as they are the only established hepatoma line assumed to be able to perform the second, as well as first, assembly step, albeit with varying efficiency depending on culture conditions [8], in regard to B-48. These studies have typically equated VLDL with whatever floated to the top of isopycnic sucrose gradients with upper limiting densities of 1.01–1.03 g/ml, a range that includes intermediate density lipoproteins (IDL) and VLDL particles much smaller than those normally found in rat plasma VLDL
Lipoproteins float towards the top at rates proportional to their flotation coefficients, and the conditions are chosen so as to distribute VLDL according to their particle size in the top 8 out of a total of 11 fractions
Summary
Dulbecco’s modified Eagle’s medium (DMEM) and all other reagents for cell culture were purchased from Life Technologies, Inc. (Gaithersburg, MD). [35S]methionine was purchased from. A 1-ml sample of human VLDL, 0.6 mg protein/ml, adjusted with NaBr to solvent density of 1.32 g/ml and containing 1% Ficoll (Sigma, St. Louis, MO) to stabilize the interface, was layered under a 10-ml linear sodium bromide gradient, density 1.25–1.30 g/ml, and centrifuged in a Beckman SW41 rotor at 18,000 rpm and 20ЊC for 90 min. Kinetic gradient ultracentrifugation of lipoproteins secreted by McA-RH7777 cells [35S]methionine-labeled lipoproteins, or unlabeled lipoproteins from 48-h conditioned media, were concentrated, brought to NaBr solvent density of 1.32 g/ml, and separated by flotation through a linear 1.25–1.30 g/ml NaBr gradient at 18,000 rpm for 90 min in an SW 41 rotor at 20ЊC, as described above for human VLDL. The cells were washed twice with in 0.2% Triton X-100/0.1% sodium sarcosylate/0.02% SDS, boiled in sample buffer containing 1% SDS, and the released apoB peptides were separated by SDS-PAGE
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