An approach to the functional analysis of lecithin-cholesterol acyltransferase. Activation by recombinant normal and mutagenized apolipoprotein AI.
Apolipoprotein AI (apo AI) of human serum high-density lipoprotein functions as an activator of lecithin-cholesterol acyltransferase (LCAT) and therefore plays an important role in reversed cholesterol transport. The mechanism of the acyltransfer, the activating polypeptide domains of apo AI and the active site of LCAT in this transesterification are not yet known. Synthetic peptides of the apo AI sequence have been designed to determine the activating structure, but did not yet lead to conclusive results. This also applies to spontaneous apo AI mutants. We therefore used the method of site-directed mutagenesis of apo AI cDNAs using the overlap extension approach by the polymerase chain reaction. These constructs were cloned into the procaryotic vector pET8c and expressed under the inducible T7 promoter. The engineered apo AI polypeptides were isolated and purified by affinity chromatography and assayed for their activator activity. The essentials of this approach to the structure and function of activators in general have successfully been exemplified for the LCAT activation by engineering apo AI mutant polypeptides a) by the deletion of two adjacent amphipathic helices (amino acid residues 146-186) and b) by introducing a point mutation (Glu111----Gln).
Bruhn
H
H
Institut für Biochemie, Medizinische Fakultät, Universität zu Köln.
Stoffel
W
W
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000032:analysis* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000032:analysis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D004269:DNA, Bacterial; D008081:Liposomes; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase