Charged Amino Acid Residues 997-1000 of Human Apolipoprotein B100 Are Critical for the Initiation of Lipoprotein Assembly and the Formation of a Stable Lipidated Primordial Particle in McA-RH7777 Cells

被引:8
|
作者
Manchekar, Medha [1 ]
Richardson, Paul E. [4 ]
Sun, Zhihuan [1 ]
Liu, Yanwen [1 ]
Segrest, Jere P. [1 ,2 ]
Dashti, Nassrin [1 ,3 ]
机构
[1] Univ Alabama Birmingham, Atherosclerosis Res Unit, Basic Sci Sect, Dept Med, Birmingham, AL 35294 USA
[2] Univ Alabama Birmingham, Med Ctr, Dept Biochem & Mol Genet, Birmingham, AL 35294 USA
[3] Univ Alabama Birmingham, Med Ctr, Dept Cell Biol, Birmingham, AL 35294 USA
[4] Coastal Carolina Univ, Dept Chem & Phys, Conway, SC 29528 USA
基金
美国国家卫生研究院;
关键词
D O I
10.1074/jbc.M804912200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We previously demonstrated that a portion, or perhaps all, of the residues between 931 and 1000 of apolipoprotein (apo) B100 are required for the initiation of apoB-containing particle assembly. Based on our structural model of the first 1000 residues of apoB (designated as apoB:1000), we hypothesized that this domain folds into a three-sided lipovitellin-like "lipid pocket" via a hairpinbridge mechanism.We proposed that salt bridges are formed between four tandem charged residues 717-720 in the turn of the hairpin bridge and four tandem complementary residues 997-1000 located at the C-terminal end of the model. To identify the specific motif within residues 931 and 1000 that is critical for apoB particle assembly, apoB:956 and apoB:986 were produced. To test the hairpin-bridge hypothesis, the following mutations were made: 1) residues 997-1000 deletion (apoB:996), 2) residues 717-720 deletion (apoB:1000 Delta 717-720), and 3) substitution of charged residues 997-1000 with alanines(apoB:996+4Ala). Characterization of particles secreted by stable transformants of McA-RH7777 cells demonstrated the following. 1) ApoB:956 did not form stable particles and was secreted as large lipid-rich aggregates. 2) ApoB:986 formed both a lipidated particle that was denser than HDL3 and large lipid-rich aggregates. 3) Compared with wild-type apoB:1000, apoB:1000 Delta 717-720 displayed the following: (i) significantly diminished capacity to form intact lipidated particles and (ii) increased propensity to form large lipid-rich aggregates. 4) Instriking contrast to wild-type apoB:1000, (i) apoB:996 and apoB:996+4Ala were highly susceptible to intracellular degradation, (ii) only a small proportion of the secreted proteins formed stable HDL3-like lipoproteins, and (iii) a majority of the secreted proteins formed large lipid-rich aggregates. We conclude that the first 1000 amino acid residues of human apoB100 are required for the initiation of nascent apoB-containing lipoprotein assembly, and residues 717-720 and 997-1000 play key roles in this process, perhaps via a hairpinbridge mechanism.
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页码:29251 / 29265
页数:15
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