Faculty Collaboration Database

Alteration in apolipoprotein A-I 22-mer repeat order results in a decrease in lecithin:cholesterol acyltransferase reactivity. J Biol Chem 1997 Mar 14;272(11):7278-84

Date

03/14/1997

Pubmed ID

9054424

DOI

10.1074/jbc.272.11.7278

Scopus ID

2-s2.0-0030906053 (requires institutional sign-in at Scopus site)   57 Citations

Abstract

Apolipoprotein A-I contains eight 22-amino acid and two 11-amino acid tandem repeats that comprise 80% of the mature protein. These repeating units are believed to be the basic motif responsible for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation. Computer analysis indicates that despite a fairly high degree of compositional similarity among the tandem repeats, significant differences in hydrophobic and amphipathic character exist. Our previous studies demonstrated that deletion of repeat 6 (143-164) or repeat 7 (165-186) resulted in a 98-99% reduction of LCAT activation as compared with wild-type apoA-I. To determine the effects of substituting one of these repeats with a more hydrophobic repeat we constructed a mutant apoA-I protein in which residues 143-164 (repeat 6) were replaced with repeat 10 (residues 220-241). The cloned mutant protein, 10F6 apoA-I, was expressed and purified from an Sf-9 cell baculoviral system and then analyzed using a number of biophysical and biochemical techniques. Recombinant complexes prepared at a 100:5:1 molar ratio of L-alpha-dimyristoylphosphatidylcholine:cholesterol:wild-type or 10F6 apoA-I showed a doublet corresponding to Stokes diameters of 114 and 108 A on nondenaturing 4-30% polyacrylamide gel electrophoresis. L-alpha-Dimyristoylphosphatidylcholine 10F6 apoA-I complexes had a 5-6-fold lower apparent Vmax/apparent Km as compared with wild-type apoA-I containing particles. As expected, monoclonal antibody epitope mapping of the lipid-free and lipid-bound 10F6 apoA-I confirmed that a domain expressed between residues 143 and 165 normally found in wild-type apoA-I was absent. The region between residues 119 and 144 in 10F6 apoA-I showed a marked reduction in monoclonal antibody binding capacity. Therefore, we speculate that the 5-6-fold lower LCAT reactivity in 10F6 compared with wild-type apoA-I recombinant particles results from increased stabilization within the 121-165 amino acid domain due to more stable apoprotein helix phospholipid interactions as well as from conformational alterations among adjacent amphipathic helix repeats.

Author List

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

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

Amino Acid Sequence
Apolipoprotein A-I
Enzyme Activation
Humans
Molecular Sequence Data
Phosphatidylcholine-Sterol O-Acyltransferase
Recombinant Proteins
Repetitive Sequences, Nucleic Acid