Structural and functional properties of the 154-171 wild-type and variant peptides of human lecithin-cholesterol acyltransferase.
The 154-171 segment of the human lecithin-cholesterol acyltransferase (LCAT) enzyme was identified as the most stable amphipathic helix in the LCAT sequence. Its mean hydrophobicity, hydrophobic moment and its orientation at a lipid/water interface are similar to those of some of the helical repeats of apolipoprotein A-IV and E. This domain was therefore proposed as a candidate peptide accounting for the association between LCAT and its lipid substrate. To investigate this hypothesis we synthesized the LCAT-(154-171)-peptide, two variants containing the natural Y156N and R158C mutations and a variant with increased hydrophobicity through Y156I, L160I, L163I and Y171W substitutions. The structural and lipid-binding properties of these synthetic peptides were investigated by turbidity, fluorescence, electron microscopy and circular dichroism. The wild-type peptide, the R158C variant in its dimeric form, as well as the more hydrophobic peptide, associated with phospholipids, whereas the Y156N and the R158C variant in its monomeric form did not. However, only the complexes generated with the hydrophobic variant were stable enough to resist dissociation during gel filtration. The wild-type peptide and hydrophobic variant formed discoidal complexes with dimyristoylglycerophosphocholine (Myr2GroPCho) as shown by negative staining electron microscopy. Comparison of the properties of the wild-type and hydrophobic variant LCAT-(154-171)-peptide stresses the contribution of the hydrophobic face of the amphipathic helix to the formation and stabilization of the peptide/lipid complexes. This is further confirmed by the decreased affinity of the Y156N variant peptide for lipids, as this mutation decreased the mean hydrophobicity of the hydrophobic face of the amphipathic helix. These results support the hypothesis that the 154-171 segment of LCAT might be involved in the interaction of the enzyme with its lipid substrate and suggest that the decreased activity of the Y156N natural LCAT mutant might result from a decreased affinity of this mutant for lipids.
Peelman
F
F
Department Biochemistry, Faculty of Medicine, University of Gent, Belgium.
Goethals
M
M
Vanloo
B
B
Labeur
C
C
Brasseur
R
R
Vandekerckhove
J
J
Rosseneu
M
M
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D005453:Fluorescence; D005454:Fluorescence Polarization; D014644:Genetic Variation; D006801:Humans; D050356:Lipid Metabolism; D008081:Liposomes; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009391:Nephelometry and Turbidimetry; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D017433:Protein Structure, Secondary; D013499:Surface Properties; D013696:Temperature; D014443:Tyrosine / Q000378:metabolism
D008081:Liposomes; D010446:Peptide Fragments; D010743:Phospholipids; D014443:Tyrosine; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D004134:Dimyristoylphosphatidylcholine