© 1995 American Heart Association, Inc.
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Characterization of Recombinant Human ApoB-48–Containing Lipoproteins in Rat Hepatoma McA-RH7777 Cells Transfected With ApoB-48 cDNA
Overexpression of ApoB-48 Decreases Synthesis of Endogenous ApoB-100
From the Departments of Pathology and Biochemistry (M.M.H., R.K.K.), the Medical College of Pennsylvania, Philadelphia, Pa; the Lipid and Lipoprotein Research Group and Department of Biochemistry (Y.Z., Z.Y.), University of Alberta, Edmonton, Canada; and COR Therapeutics (B.D.B.), South San Francisco, Calif.
Correspondence to Dr M. Mahmood Hussain, Departments of Pathology and Biochemistry, The Medical College of Pennsylvania, 2900 Queen Ln, Philadelphia, PA 19129.
Abstract We studied the effect of overexpression of apolipoprotein (apo) B-48 on the synthesis and secretion of endogenous apoB-100 in rat hepatoma McA-RH7777 cell lines stably transfected with human apoB-48 cDNA under the control of the cytomegalovirus promoter. Three cell lines that secrete 40 to 60 ng human apoB · mg cell protein-1 · h-1 were used. The recombinant human apoB-48 exhibited physicochemical characteristics (buoyant density, 1.06 to 1.21 g/mL; ß-electrophoretic mobility and diameters, 16 to 20 nm) indistinguishable from those of endogenous rat apoB-48. Overexpression of the recombinant human apoB-48 resulted in a 50% decrease in the secretion of endogenous apoB-100 but did not affect the secretion of apoE or apoA-I. Several possible mechanisms for the decreased secretion of apoB-100 were evaluated. First, recruitment of lipids into lipoproteins was shown to be unaffected since no major changes in the physicochemical properties of apoB-100–containing lipoproteins were observed. Second, the intracellular degradation of apoB-100 was not altered as the intracellular retention half-time and secretion efficiency remained unaffected by apoB-48 overexpression. Third, the posttranslational regulatory mechanisms for apoB-100 remained normal, as demonstrated by a twofold increase in apoB-100 secretion after supplementation with oleic acid. Unexpectedly, a 35% to 50% decrease in the steady-state synthesis of endogenous apoB-100 was observed in apoB-48–transfected cells compared with control cells. These data suggested that decreased secretion of apoB-100 was secondary to decreased synthesis. The decreased apoB-100 synthesis was not due to decreased steady-state levels of rat apoB-100 mRNA. These results suggest that overexpression of recombinant human apoB-48 may interfere with posttranscriptional events, possibly at the translation-translocation level, and decrease translational yield of apoB-100. These posttranscriptional events prior to the complete synthesis of the apoB-100 polypeptide can be important in the control of apoB-100 secretion.
Key Words: apoB-48 • rat hepatoma • apolipoproteins • lipoproteins • apoB-100
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