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.
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 hairpin-bridge 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:1000Delta717-720), 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 HDL(3) and large lipid-rich aggregates. 3) Compared with wild-type apoB:1000, apoB:1000Delta717-720 displayed the following: (i) significantly diminished capacity to form intact lipidated particles and (ii) increased propensity to form large lipid-rich aggregates. 4) In striking 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 HDL(3)-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 hairpin-bridge mechanism.
Manchekar
Medha
M
Department of Medicine, Basic Sciences Section, Atherosclerosis Research Unit, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294, USA.
Richardson
Paul E
PE
Sun
Zhihuan
Z
Liu
Yanwen
Y
Segrest
Jere P
JP
Dashti
Nassrin
N
D000409:Alanine / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D053299:Apolipoprotein B-100 / Q000737:chemistry*; D045744:Cell Line, Tumor; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry*; D008077:Lipoproteins, LDL / Q000737:chemistry; D008954:Models, Biological; D008957:Models, Genetic; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats
D000596:Amino Acids; D053299:Apolipoprotein B-100; D008074:Lipoproteins; D008077:Lipoproteins, LDL; D000409:Alanine