Identification of a sequence of apolipoprotein A-I associated with the activation of Lecithin:Cholesterol acyltransferase.
We aimed to distinguish between the effects of mutations in apoA-I on the requirements for the secondary structure and a specific amino acid sequence for lecithin:cholesterol acyltransferase (LCAT) activation. Several mutants were constructed targeting region 140-150: (i) two mutations affecting alpha-helical structure, deletion of amino acids 140-150 and substitution of Ala(143) for proline; (ii) two mutations not affecting alpha-helical structure, substitution of Val(149) for arginine and substitution of amino acids 63-73 for sequence 140-150; and (iii) a mutation in a similar region away from the target area, deletion of amino acids 63-73. All mutations affecting region 140-150 resulted in a 4-42-fold reduction in LCAT activation. Three mutations, apoA-I(Delta140-150), apoA-I(P143A), and apoA-I(140-150 --> 63-73), affected both the apparent V(max) and K(m), whereas the mutation apoA-I(R149V) affected only the V(max). The mutation apoA-I(Delta63-73) caused only a 5-fold increase in the K(m). All mutants, except apoA-I(P143A) and apoA-I(Delta63-73), were active in phospholipid binding assay. All mutants, except apoA-I(P143A), formed normal discoidal complexes with phospholipid. The mutation apoA-I(Delta63-73) caused a significant reduction in the stability of apoA-I.phospholipid complexes in denaturation experiments. Combined, our results strongly suggest that although the correct conformation and orientation of apoA-I in the complex with lipids are crucial for activation of LCAT, when these conditions are fulfilled, activation also strongly depends on the sequence that includes amino acids 140-150.
Sviridov
D
D
Baker Medical Research Institute, Melbourne 8008 and the Department of Biochemistry, University of Melbourne, Parkville 3052, Victoria, Australia. dmitri.Sviridov@Baker.edu.au
Hoang
A
A
Sawyer
W H
WH
Fidge
N H
NH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019791:Guanidine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
D000596:Amino Acids; D016632:Apolipoprotein A-I; D003432:Cross-Linking Reagents; D008075:Lipoproteins, HDL; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D019791:Guanidine