Importance of Central -Helices of Human Apolipoprotein A-I in the Maturation of High-Density Lipoproteins

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• 作者：Philippe G. Frank ; Duong N'Guyen ; Vivian Franklin ; Tracey Neville ; Marc Desforges ; Eric Rassart ; Daniel L. Sparks ; and Yves L. Marcel
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：September 29, 1998
• 年：1998
• 卷：37
• 期：39
• 页码：13902 - 13909
• 全文大小：85K
• 年卷期：v.37,no.39(September 29, 1998)
• ISSN：1520-4995

文摘

We have studied the role of amphipathic -helices in the ability of apoA-I to promote cholesterolefflux from human skin fibroblasts and activate lecithin:cholesterol acyltransferase (LCAT). Three apoA-Imutants were designed, each by deletion of a pair of predicted adjacent central -helices [(100-143),(122-165), (144-186)], and expressed in Escherichia coli. This strategy was used to minimizedisruption of the predicted secondary structure of the resulting protein. These three central deletion mutantshave been previously shown to be expressed as stable folded proteins but to exhibit altered phospholipid-binding properties. When recombined with phospholipids to form homogeneous LpA-I containingequivalent amounts of POPC and tested for their ability to promote diffusional cholesterol efflux fromnormal [³H]cholesterol-labeled fibroblasts, each mutant and the wild-type recombinant protein (Rec.-apoA-I) promoted cholesterol efflux with very similar rates at all the concentrations tested. Theseexperiments showed that all LpA-I could acquire cellular cholesterol with similar affinity and bindingcapacity. However, when the cell-incubated LpA-I were incubated with purified LCAT, two mutants,(122-165) and (144-186), appeared incapable of activating the enzyme. To directly determine theirability to activate LCAT, each mutant and the control were recombined with equivalent amounts ofcholesterol and phospholipid and incubated with the purified enzyme. The results show that whereasdeletion of residues 100-143 has little effect on LCAT activation, deletion of residues 122-165 or 144-186 results in an inability of the mutants to promote cholesterol esterification. In conclusion, our resultsshow that no specific sequence in the central domain of apoA-I is required for efficient diffusionalcholesterol efflux from normal fibroblasts; however, residues 144-186 appear critical for optimum LCATactivation and cholesteryl ester accumulation. Since deletion of residues 144-186 also perturbsphospholipid association and prevents the formation of large LpA-I particles [Frank, P. G., Bergeron, J.,Emmanuel, F., Lavigne, J. P., Sparks, D. L., Denèfle, P., Rassart, E., and Marcel, Y. L. (1997)Biochemistry 36, 1798-1806], the data show that this pair of -helices plays an important role in thematuration of HDL. Sequence analysis of these apoA-I helices further identifies specific residues thatappear essential to this activity.