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 r equirements 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 alp ha-helical structure, deletion of amino acids 140-150 and substitution of A la(143) for proline; (ii) two mutations not affecting alpha-helical structu re, substitution of Val(149) for arginine and substitution of amino acids 6 3-73 for sequence 140-150; and (iii) a mutation in a similar region away fr om 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(Delta 140-150), apoA-I(P143A), and apoA-I(140-150 --> 63- 73), affected both the apparent V-max and K-m, whereas the mutation apoA-I( R140V) affected only the V-max. The mutation apoA-I(Delta 63-73) caused onl y a 5-fold increase in the K-m. All mutants, except apoA-I(P143A) and apoA- I(Delta 63-73), were active in phospholipid binding assay. All mutants, exc ept apoA-I(P143A), formed normal discoidal complexes with phospholipid, The mutation apoA-I(Delta 63-73) caused a significant reduction in the stabili ty of apoA-I phospholipid complexes in denaturation experiments. Combined, our results strongly suggest that although the correct conformation and ori entation of apoA-I in the complex with lipids are crucial for activation of LCAT, when these conditions are fulfilled, activation also strongly depend s on the sequence that includes amino acids 140-150.