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 t he 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 the refore proposed as a candidate peptide accounting for the association between LCAT and its lipid substrate. To investigate this hypothesis w e synthesized the LCAT-(154-171)-peptide, two variants containing the natural Y156N and R158C mutations and a variant with increased hydroph obicity through Y156I, L1601, L1631 and Y171W substitutions. The struc tural and lipid-binding properties of these synthetic peptides were in vestigated by turbidity, fluorescence, electron microscopy and circula r dichroism. The wild-type peptide, the R158C variant in its dimeric f orm, as well as the more hydrophobic peptide, associated with phosphol ipids, whereas the Y156N and the R158C variant in its monomeric form d id not. However, only the complexes generated with the hydrophobic var iant were stable enough to resist dissociation during gel filtration. The wild-type peptide and hydrophobic variant formed discoidal complex es with dimyristoylglycerophosphocholine (Myr(2)GroPCho) as shown by n egative 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 t he formation and stabilization of the peptide/lipid complexes. This is further confirmed by the decreased affinity of the Y156N variant pept ide for lipids, as this mutation decreased the mean hydrophobicity of the hy drophobic face of the amphipathic helix. These results support the hypothesis that the 154-171 segment of LCAT might be involved in t he 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.