Abstract
Familial hypobetalipoproteinemia (FHBL) is associated with mutations in the APOB gene. We reported the first missense APOB mutation, R463W, in an FHBL kindred (Burnett, J. R., Shan, J., Miskie, B. A., Whitfield, A. J., Yuan, J., Tran, K., Mc-Knight, C. J., Hegele, R. A., and Yao, Z. (2003) J. Biol. Chem. 278, 13442-13452). Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n = 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n = 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the alpha-helical domain within the N terminus of apoB. Thus, proper folding of the alpha-helical domain of apoB-100 is essential for efficient secretion.
Highlights
Apolipoprotein6 B, a large amphipathic glycoprotein, plays a central role in human lipoprotein metabolism [1, 2]
ApoB-100 is synthesized in the liver and is an essential structural component of very low density lipoprotein (VLDL) and its metabolic products, intermediate density lipoprotein (IDL) and low density lipoprotein (LDL), and is a ligand for the LDL receptor
The presence of reduced plasma concentrations of apoB-containing lipoproteins in first degree relatives confirmed the diagnosis of heterozygous Familial hypobetalipoproteinemia (FHBL) in the proband (Fig. 1A)
Summary
Phenotyping and Genotyping of the L343V Kindred—The proband (Fig. 1A, subject II:1) was referred to a lipid disorders clinic with marked hypocholesterolemia detected on routine lipid screening. Expression of apoBs was confirmed by immunoblot analysis of conditioned media (DMEM containing 20% FBS and 0.4 mM oleate) that had been incubated with cells stably or transiently transfected with respective apoB expression plasmids. The 35S-apoB was recovered from cell and media at each chase time point by immunoprecipitation, resolved by SDS-PAGE (3–15% gel), and subjected to fluorography as described previously [35]. The samples were incubated for additional 16 h (4 °C) and diluted to 0.1% SDS with RIPA buffer, and apoB proteins were immunoprecipitated with the rabbit anti-human apoB antiserum as described above. Immunoblotting for human apoB (1D1), protein-disulfide isomerase (SPA-891, StressGen, Ann Arbor, MI), Hsp (SPA820, StressGen), p97 (RDI-PRO6527, Research Diagnostics, Inc., Concord, MA), and the N-terminal epitope of calnexin (SPA-865, StressGen) was performed using respective antibodies. Plasma lipid and apolipoprotein concentrations and APOE genotype in the L343V kindred
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