Faculty Collaboration Database

The hydrophobic face orientation of apolipoprotein A-I amphipathic helix domain 143-164 regulates lecithin:cholesterol acyltransferase activation. J Biol Chem 1998 May 08;273(19):11776-82

Date

06/13/1998

Pubmed ID

9565601

DOI

10.1074/jbc.273.19.11776

Scopus ID

2-s2.0-0032496221 (requires institutional sign-in at Scopus site)   72 Citations

Abstract

Apolipoprotein A-I (apoA-I) activates the plasma enzyme lecithin:cholesterol acyltransferase (LCAT), catalyzing the rapid conversion of lipoprotein cholesterol to cholesterol ester. Structural mutants of apoA-I have been used to study the details of apoA-I-LCAT-catalyzed cholesterol 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 examined how the orientation of the hydrophobic face, independent of an increase in overall hydrophobicity, affects LCAT activation. We designed, expressed, and characterized a mutant, reverse of 6 apoA-I (RO6 apoA-I), in which the primary amino acid sequence of repeat 6 (amino acids 143-164) was reversed from its normal orientation. This mutation rotates the hydrophobic face of repeat 6 approximately 80 degrees. Lipid-free RO6 apoA-I showed a marked stabilization when denatured by guanidine hydrochloride, but showed significant destabilization to guanidine hydrochloride denaturation in the lipid-bound state compared with wild-type apoA-I. Recombinant high density lipoprotein discs (rHDL) formed from RO6 apoA-I, sn-1-palmitoyl-sn-2-oleoyl phosphati-dylcholine, and cholesterol were approximately 12 A smaller than wild-type apoA-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 effective substrate for LCAT. Mapping the entire lipid-free and lipid-bound RO6 apoA-I with a series of monoclonal antibodies revealed that both the lipid-free and lipid-bound RO6 apoA-I displayed altered or absent epitopes in domains within and adjacent to repeat 6. Together, these results suggest that the proper alignment and orientation of the hydrophobic face of repeat 6 is an important determinant for maintaining and stabilizing helix-bilayer and helix-helix interactions.

Author List

Sorci-Thomas MG, Curtiss L, Parks JS, Thomas MJ, Kearns MW, Landrum M

Authors

Mary Sorci Thomas PhD Professor in the Medicine department at Medical College of Wisconsin
Michael J. Thomas PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Apolipoprotein A-I
Cholesterol Esters
Circular Dichroism
Enzyme Activation
Epitope Mapping
Lipoproteins, HDL
Phospholipids
Protein Binding
Protein Conformation
Protein Structure, Secondary
Repetitive Sequences, Nucleic Acid
Solubility
Sterol O-Acyltransferase
Structure-Activity Relationship