Abstract
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.
Highlights
ApoA-I is a key element of the reverse cholesterol transport pathway
Our results strongly suggest that the correct conformation and orientation of apoA-I in the complex with lipids are crucial for activation of lecithin:cholesterol acyltransferase (LCAT), when these conditions are fulfilled, activation strongly depends on the sequence that includes amino acids 140 –150
The sequence between amino acids 140 and 150 belongs to the central domain of apoA-I that is implicated in the ability of apoA-I to activate LCAT and that may be involved in the stimulation of efflux of intracellular cholesterol [15]
Summary
ApoA-I is a key element of the reverse cholesterol transport pathway. This pathway removes excess cholesterol from extrahepatic cells and protects the artery wall against developing atherosclerosis [1]. ApoA-I regulates the translocation of intracellular cholesterol to the plasma membrane [11, 12], promotes efflux of intracellular cholesterol [13,14,15,16], triggers signaling pathways that could be related to cholesterol efflux [17,18,19], and regulates expression of adhesion molecules [20] Many of these activities are related to the unique secondary structure of apoA-I: when bound to lipid, apoA-I consists of nine 22-mer and two 11-mer amphipathic ␣-helices spanning almost the entire length of apoA-I [21]. We show that in addition to the carboxyl-terminal end sites, a lipid-binding domain of apoA-I might reside between amino acids 63 and 73
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