Mouse Low Density Lipoprotein Research Articles

Tissue-specific Inhibition of Apolipoprotein B mRNA Editing in the Liver by Adenovirus-mediated Transfer of a Dominant Negative Mutant APOBEC-1 Leads to Increased Low Density Lipoprotein in Mice

APOBEC-1 is a catalytic subunit of an apolipoprotein B (apoB) mRNA editing enzyme complex. In humans it is expressed only in the intestine, whereas in mice it is expressed in both the liver and intestine. APOBEC-1 exists as a spontaneous homodimer (Lau, P. P., Zhu, H.-J., Baldini, A., Charnsangavej, C., and Chan, L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8522-8526). We tested the editing activity and dimerization potential of three different mouse APOBEC-1 mutants using in vitro editing activity assay and immunoprecipitation in the presence of epitope-tagged APOBEC-1. One catalytically inactive mutant, mu1 (H61K/C93S/C96S), that retains its capacity to dimerize with wild-type APOBEC-1 was found to inhibit the editing activity of the latter and was thus a dominant negative mutant. Two other inactive mutants that dimerized poorly with APOBEC-1 failed to inhibit its activity. Intravenous injection of a mu1 adenovirus, Admu1, in C57BL/6J mice in vivo resulted in liver-specific expression of mu1 mRNA. On days 4 and 9 after virus injection, endogenous hepatic apoB mRNA editing was 23.3 +/- 5.0 and 36.8 +/- 5.7%, respectively, compared with 65.3 +/- 11 and 71.3 +/- 5.2%, respectively, for luciferase adenovirus-treated animals. Plasma apoB-100 accounted for 95 and 93% of total plasma apoB in Admu1 animals on days 4 and 9, respectively, compared with 78 and 72% in luciferase adenovirus animals. Plasma cholesterol on day 9 was 98 +/- 17 mg/dl in the mu1-treated animals, substantially higher than phosphate-buffered saline-treated (57 +/- 9 mg/dl) or luciferase-treated (71 +/- 12 mg/dl) controls. Fast protein liquid chromatography analysis of mouse plasma showed that the intermediate density/low density lipoprotein fractions in the animals treated with the dominant negative mutant adenovirus were much higher than those in controls. We conclude that active APOBEC-1 functions as a dimer and its activity is inhibited by a dominant negative mutant. Furthermore, apoB mRNA editing determines the availability of apoB-100, which in turn limits the amount of intermediate density/low density lipoprotein that can be formed in mice. Liver-specific inhibition of apoB mRNA editing is an important component of any strategy to enhance the value of mice as a model for human lipoprotein metabolism.

Functional expression of human and mouse low density lipoprotein receptors in hybridomas

Though a mouse.human-human heterohybridoma, N12-16.63, secreting an antitetanus toxoid human monoclonal antibody grew well in a serum-free medium, its high producing subclone N12-69 required SSGF-I, a low density lipoprotein (LDL) from swine serum, or human-LDL (h-LDL) for growth. The growth-promoting action of SSGF-I was caused by its lipid fraction, and SSGF-I could be replaced completely with cholesterol in the presence of bovine serum albumin (BSA). Thus, cell line N12-69 is a cholesterol auxotroph of the heterohybridoma. N12-69 cells express both mouse and human LDL receptors on the cell surface in a ratio of 1:4. SSGF-I bound to both receptors with the same binding affinity, and h-LDL was also take up by the same receptors, though the affinity constant of the receptors for SSGF-I was 1.5 times stronger than that for h-LDL. The growth of N12-69 cells was completely inhibited by the addition of dextran sulfate, which is known to inhibit the binding of LDL to LDL receptors, to an SSGF-I or h-LDL containing medium but was not inhibited at all when dextran sulfate was added to a serum-free medium supplemented with cholesterol and BSA. Furthermore, an anti-human LDL receptor monoclonal antibody partially inhibited the growth of N12-69 cells in an SSGF-I or h-LDL containing medium. These findings suggest that N12-69 cells express both biologically active mouse and human LDL receptors on their cell surfaces and that SSGF-I or h-LDL is taken up by the both receptors to be utilized as a cholesterol source for the growth.