title: Role of the Arg123-Tyr166 paired helix of apolipoprotein A-I in lecithin:cholesterol acyltransferase activation.
abstract: The Arg123-Tyr166 central and Ala190-Gln243 carboxyl-terminal pairs of helices of apoA-I were substituted with the pair of helices of apoA-II, resulting in the apoA-I(Delta(Arg123-Tyr166), nablaA-II(Ser12-Ala75)) and apoA-I(Delta(Ala190-Gln243), nablaA-II(Ser12-Gln77)) chimeras, respectively. The structures of these chimeras in aqueous solution and in reconstituted high density lipoproteins (rHDL) and the lecithin:cholesterol acyltransferase (LCAT) activation properties of the rHDL were studied. Recombinant human apoA-I and the chimeras were expressed in Escherichia coli and purified from the periplasmic space. Binding of the apolipoproteins with palmitoyloleoylphosphatidylcholine was associated with a similar shift of Trp fluorescence maxima from 337 to 332 nm, from 339 to 334 nm, and from 337 to 333 nm, respectively. All rHDL had a Stokes radius of 4.8 nm and contained 2 apolipoprotein molecules/particle. Circular dichroism measurements revealed eight alpha-helices per apoA-I and per chimera molecule. The catalytic efficiencies of LCAT activation were 1.5 +/- 0.33 (mean +/- S.D.; n = 3), 0.054 +/- 0.009 (p < 0.001 versus apoA-I), and 1.3 +/- 0.32 (p = not significant versus apoA-I) nmol of cholesteryl ester/h/microM, respectively. The lower LCAT activity of the central domain chimera was due to a 27-fold reduced Vmax with unaltered Km. Binding of radiolabeled LCAT to rHDL of apoA-I and apoA-I(Delta(Arg123-Tyr166), nablaA-II(Ser12-Ala75)) was very similar. In conclusion, although substitution of the Arg123-Tyr166 central or Ala190-Gln243 carboxyl-terminal pair of helices of apoA-I with the pair of helices of apoA-II yields chimeras with structure similar to that of native apoA-I, exchange of the central domain (but not the carboxyl-terminal domain) of apoA-I reduces the rate of LCAT activity that is independent of binding to rHDL.
authors.affiliation: Center for Molecular and Vascular Biology, University of Leuven, B-3000 Leuven, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Dhoest
authors.forename: Z
authors.initials: Z
authors.lastname: Zhao
authors.forename: B
authors.initials: B
authors.lastname: De Geest
authors.forename: E
authors.initials: E
authors.lastname: Deridder
authors.forename: A
authors.initials: A
authors.lastname: Sillen
authors.forename: Y
authors.initials: Y
authors.lastname: Engelborghs
authors.forename: D
authors.initials: D
authors.lastname: Collen
authors.forename: P
authors.initials: P
authors.lastname: Holvoet
mesh_terms: D016632:Apolipoprotein A-I / Q000737:chemistry / Q000378:metabolism*; D016633:Apolipoprotein A-II / Q000737:chemistry / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism; D008970:Molecular Weight; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D008075:Lipoproteins, HDL; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D001120:Arginine; D007862:Phosphatidylcholine-Sterol O-Acyltransferase