Functional analysis of the missense APOC3 mutation Ala23Thr associated with human hypotriglyceridemia

• Published in: Journal of lipid research Vol. 51; no. 6; pp. 1524 - 1534
• Main Authors: Sundaram, Meenakshi, Zhong, Shumei, Khalil, Maroun Bou, Zhou, Hu, Jiang, Zhenghui G., Zhao, Yang, Iqbal, Jahangir, Hussain, M. Mahmood, Figeys, Daniel, Wang, Yuwei, Yao, Zemin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2010; American Society for Biochemistry and Molecular Biology; The American Society for Biochemistry and Molecular Biology; Elsevier
• Subjects: Alanine - genetics; apolipoprotein C-III; Apolipoproteins C - chemistry; Apolipoproteins C - genetics; Apolipoproteins C - metabolism; Brefeldin A - pharmacology; Carrier Proteins - metabolism; Cell Line; Gene Expression Regulation; Humans; Lipid Metabolism Disorders - genetics; Lipoproteins, IDL - metabolism; Lipoproteins, LDL - metabolism; Lipoproteins, VLDL - metabolism; lumenal lipid droplets; Microsomes - metabolism; Models, Molecular; Mutation, Missense; Protein Binding - drug effects; Protein Structure, Secondary; Threonine - genetics; triacylglycerol; Triglycerides - metabolism; VLDL assembly; VLDL assembly; triacylglycerol; apolipoprotein C-III; lumenal lipid droplets
• ISSN: 0022-2275; 1539-7262
• DOI: 10.1194/jlr.M005108

We have shown that expression of apolipoprotein (apo) C-III promotes VLDL secretion from transfected McA-RH7777 cells under lipid-rich conditions. To determine structural elements within apoC-III that confer to this function, we contrasted wild-type apoC-III with a mutant Ala23Thr originally identified in hypotriglyceridemia subjects. Although synthesis of [3H]glycerol-labeled TAG was comparable between cells expressing wild-type apoC-III (C3wt cells) or Ala23Thr mutant (C3AT cells), secretion of [3H]TAG from C3AT cells was markedly decreased. The lowered [3H]TAG secretion was associated with an inability of C3AT cells to assemble VLDL1. Moreover, [3H]TAG within the microsomal lumen in C3AT cells was 60% higher than that in C3wt cells, yet the activity of microsomal triglyceride-transfer protein in C3AT cells was not elevated. The accumulated [3H]TAG in C3AT microsomal lumen was mainly associated with lumenal IDL/LDL-like lipoproteins. Phenotypically, this [3H]TAG fractionation profiling resembled what was observed in cells treated with brefeldin A, which at low dose specifically blocked the second-step VLDL1 maturation. Furthermore, lumenal [35S]Ala23Thr protein accumulated in IDL/LDL fractions and was absent in VLDL fractions in C3AT cells. These results suggest that the presence of Ala23Thr protein in lumenal IDL/LDL particles might prevent effective fusion between lipid droplets and VLDL precursors. Thus, the current study reveals an important structural element residing within the N-terminal region of apoC-III that governs the second step VLDL1 maturation.