Identification of a Sequence of Apolipoprotein A-I Associated with the Activation of Lecithin:Cholesterol Acyltransferase

Dmitri Sviridov, Anh Hoang, William H. Sawyer, Noel H. Fidge
2000 Journal of Biological Chemistry  
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 ␣-helical structure, deletion of amino acids 140 -150 and substitution of Ala 143 for proline; (ii) two mutations not affecting ␣-helical structure, substitution of Val 149 for arginine and
more » ... 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(⌬140 -150), apoA-I(P143A), and apoA-I(140 -150 3 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(⌬63-73) caused only a 5-fold increase in the K m . All mutants, except apoA-I(P143A) and apoA-I(⌬63-73), were active in phospholipid binding assay. All mutants, except apoA-I(P143A), formed normal discoidal complexes with phospholipid. The mutation apoA-I(⌬63-73) caused a significant reduction in the stability of apoA-I⅐phospholipid 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.
doi:10.1074/jbc.m000962200 pmid:10781581 fatcat:llt2ipmexjdndcm2aamvc3sxfu