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J. Biol. Chem., Vol. 283, Issue 43, 29251-29265, October 24, 2008

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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^*

Medha Manchekar, Paul E. Richardson, Zhihuan Sun, Yanwen Liu, Jere P. Segrest^¶, and Nassrin Dashti^||1

From the Department of Medicine, Basic Sciences Section, Atherosclerosis Research Unit, ^||Department of Cell Biology, and ^¶Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294 and the Department of Chemistry and Physics, Coastal Carolina University, Conway, South Carolina 29528

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:1000717–720), 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:1000717–720 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 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 hairpin-bridge mechanism.

Received for publication, June 27, 2008 , and in revised form, August 22, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants HL084685 and PO1 HL34343. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Medicine, University of Alabama at Birmingham, 1808 7th Ave. South, BDB-D680, Birmingham, AL 35292-0012. Tel.: 205-975-2159; Fax: 205-975-8079; E-mail: ndashti{at}uab.edu.

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