Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound states
Val156 of apolipoprotein A-I (apoA-I) was found to be a key amino acid in the structure and function of high density lipoprotein (HDL) (J. Biol. Chem., 275: 26821–26827, 2000). To determine more precisely the functions of the individual amino acids proximal to Val156, serial point mutants of proapoA...
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| Format: | Article |
| Language: | English |
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Elsevier
2005-03-01
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| Series: | Journal of Lipid Research |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520340438 |
| _version_ | 1819072659662569472 |
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| author | Jong-Min Han Tae-Sook Jeong Woo Song Lee Inho Choi Kyung-Hyun Cho |
| author_facet | Jong-Min Han Tae-Sook Jeong Woo Song Lee Inho Choi Kyung-Hyun Cho |
| author_sort | Jong-Min Han |
| collection | DOAJ |
| description | Val156 of apolipoprotein A-I (apoA-I) was found to be a key amino acid in the structure and function of high density lipoprotein (HDL) (J. Biol. Chem., 275: 26821–26827, 2000). To determine more precisely the functions of the individual amino acids proximal to Val156, serial point mutants of proapoA-I, including V156K, D157K, and A158E, were overexpressed and purified to at least 95% purity. In the lipid-free state, A158E exhibited the most profound self-associative patterns and the least pronounced dimyristoyl phosphatidylcholine (DMPC) clearance activities. In the lipid-bound state, A158E formed a larger reconstituted HDL (rHDL) with palmitoyloleoyl phosphatidylcholine (POPC), ∼120 Å, whereas other mutants and the wild type (WT) formed 97 Å of POPC-rHDL. Cross-linking analysis revealed that A158E-rHDL harbored at least four protein molecules in the particle, while other rHDL conformations contained only two protein molecules. All of the POPC-rHDL produced smaller HDL, around 78 Å, after 24 h of incubation in the presence of low density lipoprotein at 37°C. V156K and A158E exhibited decreased lecithin:cholesterol acyltransferase activation activity in the POPC-rHDL state, showing <2% of WT reactivity (apparent Vmax/Km). A158E also displayed markedly different properties in secondary structure, and its accessibility to proteolytic enzymes is different.These results suggest that the two amino acids in helix 6, Val156 and Ala158, are critical to both the structure and function of rHDL. |
| first_indexed | 2024-12-21T17:41:15Z |
| format | Article |
| id | doaj.art-6dca1584b9ec4269801f62e77634d6a2 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-21T17:41:15Z |
| publishDate | 2005-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-6dca1584b9ec4269801f62e77634d6a22022-12-21T18:55:36ZengElsevierJournal of Lipid Research0022-22752005-03-01463589596Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound statesJong-Min Han0Tae-Sook Jeong1Woo Song Lee2Inho Choi3Kyung-Hyun Cho4National Research Laboratory of Lipid Metabolism and Atherosclerosis, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, 305-333, South Korea; School of Biotechnology and School of Biological Resources, Yeungnam University, Kyungsan, 712-749, South KoreaNational Research Laboratory of Lipid Metabolism and Atherosclerosis, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, 305-333, South Korea; School of Biotechnology and School of Biological Resources, Yeungnam University, Kyungsan, 712-749, South KoreaNational Research Laboratory of Lipid Metabolism and Atherosclerosis, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, 305-333, South Korea; School of Biotechnology and School of Biological Resources, Yeungnam University, Kyungsan, 712-749, South KoreaNational Research Laboratory of Lipid Metabolism and Atherosclerosis, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, 305-333, South Korea; School of Biotechnology and School of Biological Resources, Yeungnam University, Kyungsan, 712-749, South KoreaNational Research Laboratory of Lipid Metabolism and Atherosclerosis, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, 305-333, South Korea; School of Biotechnology and School of Biological Resources, Yeungnam University, Kyungsan, 712-749, South KoreaVal156 of apolipoprotein A-I (apoA-I) was found to be a key amino acid in the structure and function of high density lipoprotein (HDL) (J. Biol. Chem., 275: 26821–26827, 2000). To determine more precisely the functions of the individual amino acids proximal to Val156, serial point mutants of proapoA-I, including V156K, D157K, and A158E, were overexpressed and purified to at least 95% purity. In the lipid-free state, A158E exhibited the most profound self-associative patterns and the least pronounced dimyristoyl phosphatidylcholine (DMPC) clearance activities. In the lipid-bound state, A158E formed a larger reconstituted HDL (rHDL) with palmitoyloleoyl phosphatidylcholine (POPC), ∼120 Å, whereas other mutants and the wild type (WT) formed 97 Å of POPC-rHDL. Cross-linking analysis revealed that A158E-rHDL harbored at least four protein molecules in the particle, while other rHDL conformations contained only two protein molecules. All of the POPC-rHDL produced smaller HDL, around 78 Å, after 24 h of incubation in the presence of low density lipoprotein at 37°C. V156K and A158E exhibited decreased lecithin:cholesterol acyltransferase activation activity in the POPC-rHDL state, showing <2% of WT reactivity (apparent Vmax/Km). A158E also displayed markedly different properties in secondary structure, and its accessibility to proteolytic enzymes is different.These results suggest that the two amino acids in helix 6, Val156 and Ala158, are critical to both the structure and function of rHDL.http://www.sciencedirect.com/science/article/pii/S0022227520340438apoA-I mutantsreconstituted HDLlecithin:cholesterol acyltransferasestructure-functionmiddle mobile region |
| spellingShingle | Jong-Min Han Tae-Sook Jeong Woo Song Lee Inho Choi Kyung-Hyun Cho Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound states Journal of Lipid Research apoA-I mutants reconstituted HDL lecithin:cholesterol acyltransferase structure-function middle mobile region |
| title | Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound states |
| title_full | Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound states |
| title_fullStr | Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound states |
| title_full_unstemmed | Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound states |
| title_short | Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound states |
| title_sort | structural and functional properties of v156k and a158e mutants of apolipoprotein a i in the lipid free and lipid bound states |
| topic | apoA-I mutants reconstituted HDL lecithin:cholesterol acyltransferase structure-function middle mobile region |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520340438 |
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