THE HYDROPHOBIC FACE ORIENTATION OF APOLIPOPROTEIN-A-I AMPHIPATHIC HELIX DOMAIN 143-164 REGULATES LECITHIN-CHOLESTEROL ACYLTRANSFERASE ACTIVATION

Apolipoprotein A-I (apoA-I) activates the plasma enzyme lecithin:chole sterol acyltransferase (LCAT), catalyzing the rapid conversion of lipo protein cholesterol to cholesterol ester, Structural mutants of apoA-I have been used to study the details of apoA-I-LCAT-catalyzed choleste rol ester formation. Several studies have shown that the alpha-helical segments corresponding to amino acids 143-164 and 165-186 (repeats 6 and 7) are essential for LCAT activation. In the present studies, we e xamined how the orientation of the hydrophobic face, independent of an increase in overall hydrophobicity, affects LCAT activation. we desig ned, expressed, and characterized a mutant, reverse of 6 apoA-I (RO6 a poA-I), in which the primary amino acid sequence of repeat 6 (amino ac ids 143-164) was reversed from its normal orientation. This mutation r otates the hydrophobic face of repeat 6 approximately 80 degrees, Lipi d-free RO6 apoA-I showed a marked stabilization when denatured by guan idine hydrochloride, but showed significant destabilization to guanidi ne hydrochloride denaturation in the Lipid-bound state compared with w ild-type apoA-I, Recombinant high density lipoprotein discs (rHDL) for med from RO6 apoA-I, sn-1-palmitoyl-sn-2-oleoyl phosphatidylcholine, a nd cholesterol were approximately 12 Angstrom smaller than wild-type a poA-I rHDL, The reduced size suggests that one of the repeats did not effectively participate in phospholipid binding and organization. The sn-1-palmitoyl-sn-2-oleoyl phosphatidylcholine RO6 rHDL were a less ef fective substrate for LCAT, Mapping the entire lipid-free and Lipid-bo und RO6 apoA-I with a series of monoclonal antibodies revealed that bo th the lipid-free and lipid-bound RO6 apoA-I displayed altered or abse nt epitopes in domains within and adjacent to repeat 6, Together, thes e results suggest that the proper alignment and orientation of the hyd rophobic face of repeat 6 is an important determinant for maintaining and stabilizing helix-bilayer and helix-helix interactions.