Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements.
The central region of apolipoprotein A-I (apoA-I), spanning residues 143--165, has been implicated in lecithin:cholesterol acyltransferase (LCAT) activation and also in high density lipoprotein (HDL) structural rearrangements. To examine the role of individual amino acids in these functions, we constructed, overexpressed, and purified two additional point mutants of apoA-I (P143R and R160L) and compared them with the previously studied V156E mutant. These mutants have been reported to occur naturally and to affect HDL cholesterol levels and cholesterol esterification in plasma. The P143R and R160L mutants were effectively expressed in Escherichia coli as fusion proteins and were isolated in at least 95% purity. In the lipid-free state, the mutants self-associated similarly to wild-type protein. All the mutants, including V156E, were able to lyse dimyristoylphosphatidylcholine liposomes. In the lipid-bound state, the major reconstituted HDL (rHDL) of the mutants had diameters similar to wild type (96--98 A). Circular dichroism and fluorescence methods revealed no major differences among the structures of the lipid-free or lipid-bound mutants and wild type. In contrast, the V156E mutant had exhibited significant structural, stability, and self-association differences compared with wild-type apoA-I in the lipid-free state, and formed rHDL particles with larger diameters. In this study, limited proteolytic digestion with chymotrypsin showed that the V156E mutant, in lipid-free form, has a distinct digestion pattern and surface exposure of the central region, compared with wild type and the other mutants. Reactivity of rHDL with LCAT was highest for wild type (100%), followed by P143R (39%) and R160L (0.6%). Tested for their ability to rearrange into 78-A particles, the rHDL of the two mutants (P143R and R160L) behaved normally, compared with the rHDL of V156E, which showed no rearrangement after the 24-h incubation with low density lipoprotein (LDL). Similarly, the rHDL of V156E was resistant to rearrangement in the presence of apoA-I or apoA-II. These results indicate that structural changes are absent or modest for the P143R and R160L mutants, especially in rHDL form; that these mutants have normal conformational adaptability; and that LCAT activation is obliterated for R160L.Thus, individual amino acid changes may have markedly different structural and functional consequences in the 143--165 region of apoA-I. The R160L mutation appears to have a direct effect in LCAT activation, while the P143R mutation results in only minor structural and functional effects. Also, the processes for LCAT activation and hinge mobility appear to be distinct even if the same region of apoA-I is involved. -- Cho, K-H., D. M. Durbin, and A. Jonas. Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements. J. Lipid Res. 2001. 42: 379--389.
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
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D000596:Amino Acids / Q000502:physiology*; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D008075:Lipoproteins, HDL / Q000737:chemistry*; D008081:Liposomes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D010713:Phosphatidylcholines; D011487:Protein Conformation; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D000596:Amino Acids; D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D008081:Liposomes; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D002918:Chymotrypsin; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; D004134:Dimyristoylphosphatidylcholine