Secretion from cell culture of HDL and VLDL bearing apoB-33 with a large internal deletion

Wu, Ming-Jiuan; Chen-Liu, L W; Xiao, Qunong; Phillips, Martin; Elovson, John; Linton, MacRae F.; Young, Stephen G.; Schumaker, Verne N.

doi:10.1016/s0022-2275(20)30032-8

Cited by 4 publications

(7 citation statements)

References 30 publications

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“…We have recently reported that a McA-RH7777 permanent cell line expressing B-18/95 (a fusion of apoB-18 with the C-terminal 80-95% of the human apoB sequence) secreted significant amounts of this peptide as VLDL (29). How could this phenomenon be reconciled with our present findings?…”

Section: Discussionsupporting

confidence: 77%

“…It should be pointed out that there is no assurance that even the 2-5% of the various human apoB peptides in our experiments that appeared in the VLDL region of the gradient did so as the result of having undergone true second-step VLDL assembly. Elimination of the trivial alter-native explanation, i.e., their "piggy-backing" by nonspecific adsorption to endogenous rat apoB-containing VLDL, requires demonstration by species-specific immuno-absorption that the rat and human apoB peptides reside on separate particles (29). Although we have previously demonstrated this to be the case for a different human apoB construct (29), the amounts of materials available in the present study were insufficient for such analysis.…”

Section: Discussionmentioning

confidence: 70%

“…McA-RH7777 cells (ATCC CRL 1601) were obtained from the American Type Culture Collection (Rockville, MD). McA-RH7777 cells stably transformed with pB31 and pB53 (referred to as B31 and B53, respectively) were previously described (29). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum, re-fed every other day, and split 1:20 in the same medium every 7 days.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

“…The pulse medium was collected into 10 m m EDTA, 0.5% sodium azide, 10 m m phenylmethylsulfonyl fluoride (PMSF), 10 g/ml trypsin inhibitor, 40 g/ml aprotinin, 10 g/ml leupeptin, 10 g/ml pepstatin A, and 1.92 g/ml ALLN. Total lipoproteins were isolated from the media as described (29), and subjected to kinetic gradient ultracentrifugation.…”

Section: Metabolic Labelingmentioning

confidence: 99%

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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

Journal of Lipid Research

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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.

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Section: Discussionsupporting

confidence: 77%

Section: Discussionmentioning

confidence: 70%

Section: Cell Culture and Transfectionmentioning

confidence: 99%

Section: Metabolic Labelingmentioning

confidence: 99%

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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

Journal of Lipid Research

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“…An inverse correlation between the length of apoB and the density of secreted apoB-containing lipoproteins has been observed in the plasma of patients with HBLP (4,5). The decrease in particle density, due to increased association with lipids of the longer truncated apoB forms, has also been observed in in vitro studies in human (HepG2) (12)(13)(14)(15)(16) and rat hepatoma-derived cells (13,(17)(18)(19)(20) transfected with human cDNAs encoding various lengths of apoB (21).…”

mentioning

confidence: 93%

Specificity of Lipid Incorporation Is Determined by Sequences in the N-Terminal 37 of ApoB

et al. 2000

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The N-terminal 17% of apolipoprotein B (apoB-17) is secreted lipid-poor while apoB-41 particles are secreted with a triacylglycerol (TAG)-rich core. Thus, the sequence between apoB-17 and apoB-41 is necessary for the assembly of TAG-rich lipoproteins. To delineate this region, C127 cells were permanently transfected to secrete the N-terminal 29, 32.5, or 37% of apoB. Density gradient centrifugation showed that secreted apoB-29, apoB-32.5, and apoB-37 had peak densities of 1.25, 1.22, and 1.16 g/mL and percent lipid of particle weights of 30, 37, and 49%, respectively. Calculated anhydrous particle diameters were: apoB-29 = 81 A, apoB-32.5 = 88 A, and apoB-37 = 101 A. Immunoprecipitated particles labeled with [(3)H]oleate showed that, as apoB length increased from apoB-29 to apoB-32.5 and apoB-37, the number of TAG (core) molecules per apoB particle increased almost 16-fold from 8 to 32 to 124, while phospholipids and diacylglycerols (surface lipids) increased only slightly from 71 to 87 to 97 molecules, respectively. Thus, sequences in the C-terminus of apoB-29 bind phospholipids and diacylglycerols, sequences between apoB-29 and apoB-32.5 augment TAG binding and sequences between apoB-32.5 and apoB-41 account for the marked incorporation of TAG at a rate of approximately 1 TAG per 2 amino acids. Cryoelectron micrographs of isolated apoB-37 particles revealed mostly spherical particles of approximately 110 A (11.0 nm) with an electron lucent center, consistent with these particles having a TAG core. We suggest that the predicted amphipathic beta-sheets beginning at apoB-29, starts to preferentially recruit core lipids into apoB and propose that the consistent presence of DAG in the secreted particles may have a role in fission of the nascent lipoprotein particles from the endoplasmic reticulum membrane.

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Targeting PCSK9 for therapeutic gains: Have we addressed all the concerns?

et al. 2016

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Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) regulates the expression of low-density lipoprotein (LDL)-receptors, through reducing their recycling by binding to the receptor along with LDL and targeting it for lysosomal destruction. PCSK9 also enhances the degradation of very-low-density-lipoprotein receptor (VLDLR) and lipoprotein receptor-related protein 1 (LRP-1) in a LDL-receptor independent manner. This role in lipid homeostasis presents PCSK9 as an attractive target for the therapeutic management of familial hypercholesterolemia as well as other refractory dyslipidaemias. However, PCSK9 mediates multifarious functions independent of its role in lipid homeostasis, which can be grouped under "pleiotropic functions" of the protein. This includes PCSK9's role in: trafficking of epithelial sodium channel; hepatic regeneration; pancreatic integrity and glucose homeostasis; antiviral activity; antimalarial activity; regulation of different cell signalling pathways; cortical neural differentiation; neuronal apoptosis and Alzheimer's disease. The question that needs to be investigated in depth is "How will the pleotropic functions of PCSK9, be affected by the therapeutic intervention of the protease's LDL-receptor lowering activity?" In this review, we appraise the different lipid lowering strategies targeting PCSK9 in light of the protein's different pleiotropic functions. Additionally, we delineate the key areas that require further examination, to ensure the long-term safety of the above lipid-lowering strategies.

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Secretion from cell culture of HDL and VLDL bearing apoB-33 with a large internal deletion

Cited by 4 publications

References 30 publications

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

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

Specificity of Lipid Incorporation Is Determined by Sequences in the N-Terminal 37 of ApoB

Targeting PCSK9 for therapeutic gains: Have we addressed all the concerns?

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