Alteration in apolipoprotein A-I 22-mer repeat order results in a decrease in lecithin:cholesterol acyltransferase reactivity.
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.
Sorci-Thomas
M G
MG
Department of Comparative Medicine, La Jolla, California 92034, USA. msthomas@isnet.is.bgsm.edu
Curtiss
L
L
Parks
J S
JS
Thomas
M J
MJ
Kearns
M W
MW
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid
D016632:Apolipoprotein A-I; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase