Faculty Collaboration Database

Apolipoprotein A-I helix 6 negatively charged residues attenuate lecithin-cholesterol acyltransferase (LCAT) reactivity. Biochemistry 2005 Apr 12;44(14):5409-19

Date

04/06/2005

Pubmed ID

15807534

DOI

10.1021/bi047412v

Scopus ID

2-s2.0-16844370698 (requires institutional sign-in at Scopus site)   32 Citations

Abstract

Apolipoprotein A-I (apoA-I), the major protein in high density lipoprotein (HDL) regulates cholesterol homeostasis and is protective against atherosclerosis. An examination of the amino acid sequence of apoA-I among 21 species shows a high conservation of positively and negatively charged residues within helix 6, a domain responsible for regulating the rate of cholesterol esterification in plasma. These observations prompted an investigation to determine if charged residues in helix 6 maintain a structural conformation for protein-protein interaction with lecithin-cholesterol acyltransferase (LCAT) the enzyme for which apoA-I acts as a cofactor. Three apoA-I mutants were engineered; the first, (3)/(4) no negative apoA-I, eliminated 3 of the 4 negatively charged residues in helix 6, no negative apoA-I (NN apoA-I) eliminated all four negative charges, while all negative (AN apoA-I) doubled the negative charge. Reconstituted phospholipid-containing HDL (rHDL) of two discrete sizes and compositions were prepared and tested. Results showed that LCAT activation was largely influenced by both rHDL particle size and the net negative charge on helix 6. The 80 A diameter rHDL showed a 12-fold lower LCAT catalytic efficiency when compared to 96 A diameter rHDL, apparently resulting from an increased protein-protein interaction, at the expense of lipid-protein association on the 80 A rHDL. When mutant apoproteins were compared bound to the two different sized rHDL, a strong inverse correlation (r = 0.85) was found between LCAT catalytic efficiency and apoA-I helix 6 net negative charge. These results support the concept that highly conserved negatively charged residues in apoA-I helix 6 interact directly and attenuate LCAT activation, independent of the overall particle charge.

Author List

Alexander ET, Bhat S, Thomas MJ, Weinberg RB, Cook VR, Bharadwaj MS, Sorci-Thomas 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
Base Sequence
DNA Primers
Electrophoresis, Agar Gel
Kinetics
Particle Size
Phosphatidylcholine-Sterol O-Acyltransferase