Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships. J Biol Chem 1993 Oct 05;268(28):21403-9
Date
10/05/1993Pubmed ID
8407982DOI
10.1016/s0021-9258(19)36938-8Scopus ID
2-s2.0-0027384540 (requires institutional sign-in at Scopus site) 120 CitationsAbstract
A series of mutant apolipoprotein (apoA-I) constructs were designed and then expressed in cell culture to identify structural domains within the mature native apoA-I protein that participate in the activation of the plasma enzyme, lecithin-cholesterol acyltransferase (LCAT). Evolutionary conservation analysis has shown previously that apoA-I contains eight repeats containing 22 amino acids and two repeats containing 11 amino acids that are highly conserved among species as well as within the apolipoprotein supergene family. These tandem repeats begin at residue 44 and are usually marked by a proline residue, with six of the 22-mer repeats showing high amphipathic alpha-helical character. To determine if specific 11- or 22-amino acid domains are essential for maximal LCAT activation within the entire native protein, each of the 10 repeats was sequentially deleted using a polymerase chain reaction based method of mutagenesis. The wild-type and mutant apoA-I gene constructs were expressed in Chinese hamster ovary (CHO) cells and stable lines established. Wild-type and mutant apoA-I protein were purified from 48-96-h conditioned serum-free medium and characterized by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Wild-type apoA-I showed a single migrating band of 28,000 daltons that corresponded to the mobility of human plasma apoA-I, whereas apoA-I deletion mutants (lacking 22- or 11-mer repeats) showed the corresponding shift to lower molecular size. To measure the relative LCAT activation of all deletion mutant apoA-I proteins relative to wild-type apoA-I, an assay system utilizing small unilamellar vesicles as the lipid substrate was used. The results of these studies suggest that several central amphipathic alpha-helical regions within the mature protein are critical in LCAT activation.
Author List
Sorci-Thomas M, Kearns MW, Lee JPAuthor
Mary Sorci Thomas PhD Professor in the Medicine department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsApolipoprotein A-I
Base Sequence
CHO Cells
Cricetinae
Enzyme Activation
Humans
Molecular Sequence Data
RNA, Messenger
Sterol O-Acyltransferase
Structure-Activity Relationship
