Reconstitution of the glycosylphosphatidylinositol-anchored protein Thy-1: interaction with membrane phospholipids and galactosylceramide.
Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are proposed to interact preferentially with glycosphingolipids and cholesterol to form microdomains, which may play an important role in apical targeting and signal transduction. The objective of the present study was to investigate the interaction of the GPI-anchored protein Thy-1 with phospholipids and a glycosphingolipid. Purified Thy-1 was reconstituted into lipid bilayer vesicles of dimyristoyl-phosphatidylcholine (DMPC) alone or in combination with galactosylceramide (GC). The ability of Thy-1 to perturb the gel to a liquid-crystalline phase transition of DMPC was examined by differential scanning calorimetry. As the mole fraction of Thy-1 increased, the phase transition enthalpy, deltaH, declined. Analysis indicated that each molecule of Thy-1 perturbed over 50 phospholipids, suggesting that, in addition to the anchor insertion into the bilayer, the protein itself may interact with the membrane surface. Inclusion of 5% w/w GC in the bilayer resulted in a striking change in the interaction of Thy-1 with phospholipids. At low Thy-1 content, there was a reduction in the phase transition temperature and an increase in phospholipid cooperativity, suggesting the formation of Thy-1/GC-enriched domains. DeltaH initially decreased with increasing Thy-1 content of the bilayer; however, at higher Thy-1 mole ratios, deltaH rose again. These results are interpreted in terms of a model whereby, at low protein:lipid mole ratios, Thy-1 preferentially sequesters GC to form enriched microdomains. At high protein:lipid mole ratios, Thy-1 may alter its conformation in response to steric crowding within these domains such that its interaction with the bilayer surface is reduced.
Reid-Taylor
K L
KL
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, ON, Canada.
Chu
J W
JW
Sharom
F J
FJ
D015153:Blotting, Western; D002152:Calorimetry, Differential Scanning; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005699:Galactosylceramides / Q000737:chemistry*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000302:isolation & purification; D008563:Membrane Lipids / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*
D005699:Galactosylceramides; D017261:Glycosylphosphatidylinositols; D008563:Membrane Lipids; D010743:Phospholipids