Structure-function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis.
To investigate structure and function relations of a new member of the exchangeable apolipoprotein family that modulates plasma lipid levels, recombinant human apolipoprotein (apo) A-V was produced in Escherichia coli and isolated by a combination of nickel chelation affinity chromatography and reversed-phase HPLC. Antibodies directed against apoA-V were generated and employed in immunoblotting experiments. Anti-apoA-V IgG gave a strong response against recombinant apoA-V from E. coli and human apoA-V expressed in transgenic mice, but did not recognize human apoA-I or apoA-IV. In neutral-pH buffers, at concentrations of >0.1 mg/mL, isolated lipid-free apoA-V is poorly soluble. By contrast, apoA-V is soluble in 50 mM sodium citrate (pH 3.0). Far-UV circular dichroism analysis and spectral deconvolution reveal that apoA-V possesses 32% alpha-helix, 33% beta-sheet, 16% beta-turn, and 18% random coil secondary structure conformers. Temperature-induced denaturation studies gave rise to a transition midpoint of 47.1 degrees C. Upon being cooled to ambient temperature from 85 degrees C, apoA-V failed to recover all of the negative ellipticity present in unheated apoA-V. ApoA-V interacts with bilayer vesicles of dimyristoylphosphatidylcholine to form discoidal complexes with diameters in the range of 15-20 nm. However, apoA-V was a poor activator of lecithin:cholesterol acyltransferase where the activity was 8.5 +/- 1.8% of that of apoA-I. Furthermore, apoA-V failed to support enhanced efflux of cholesterol from cAMP-treated J774 macrophages, although low levels of efflux were obtained from unstimulated cells. Taken together, the results demonstrate recombinant apoA-V possesses unique structural and functional characteristics, in keeping with its proposed role in the modulation of plasma lipid levels.
Beckstead
Jennifer A
JA
Lipid Biology in Health and Disease Research Group, Children's Hospital Oakland Research Institute, Oakland, California 94609, USA.
Oda
Michael N
MN
Martin
Dale D O
DD
Forte
Trudy M
TM
Bielicki
John K
JK
Berger
Trish
T
Luty
Robert
R
Kay
Cyril M
CM
Ryan
Robert O
RO
D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism; D000072040:Apolipoprotein A-V; D001053:Apolipoproteins; D001054:Apolipoproteins A / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002784:Cholesterol / Q000378:metabolism; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D006041:Goats; D006706:Homeostasis; D006801:Humans; D015151:Immunoblotting; D008051:Lipid Bilayers / Q000378:metabolism; D008055:Lipids / Q000097:blood*; D051379:Mice; D008822:Mice, Transgenic; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
C437704:APOA5 protein, human; C499058:Apoa5 protein, mouse; D016632:Apolipoprotein A-I; D000072040:Apolipoprotein A-V; D001053:Apolipoproteins; D001054:Apolipoproteins A; D008051:Lipid Bilayers; D008055:Lipids; D011994:Recombinant Proteins; C022205:apolipoprotein A-IV; D002784:Cholesterol; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D005982:Glutathione Transferase; D004134:Dimyristoylphosphatidylcholine