Identification of specific amphipathic alpha-helical sequence of human apolipoprotein A-IV involved in lecithin:cholesterol acyltransferase activation.
To investigate the structure-function relationship of human apolipoprotein A-IV (apoA-IV), several deletion mutants of this protein were constructed by sequentially removing pairs of 22-residue repeats, potentially having an amphipathic alpha-helical conformation. The mutants, produced as recombinant poly-histidine-tagged apolipoproteins (t-apo) in Escherichia coli, assembled with phosphatidylcholine (i.e. dimyristoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, or egg lecithin) as did native apoA-IV. Lecithin:cholesterol acyltransferase (LCAT) cofactor function, measured as cholesterol esterification occurring when t-apo-phosphatidylcholine-cholesterol complexes were incubated with purified enzyme, decreased significantly when pairs of repeats between residues 117 and 248 were deleted and most markedly when residues 117-160 were deleted. LCAT cofactor activity decreased by 90 and 75%, respectively, when egg lecithin or palmitoyloleoylphosphatidylcholine was used to form the particles with the delta aa 117-160 mutant. Thus, on the basis of deletion scanning of t-apo, residues 117-160 seem to be involved in the LCAT cofactor function of apoA-IV.
Emmanuel
F
F
Biotechnology Department, Rhône-Poulenc Rorer S.A., France.
Steinmetz
A
A
Rosseneu
M
M
Brasseur
R
R
Gosselet
N
N
Attenot
F
F
Cuiné
S
S
Séguret
S
S
Latta
M
M
Fruchart
J C
JC
D000595:Amino Acid Sequence; D001054:Apolipoproteins A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D011487:Protein Conformation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D001054:Apolipoproteins A; C022205:apolipoprotein A-IV; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D004134:Dimyristoylphosphatidylcholine