Measurement of the anchorage force between GPI-anchored alkaline phosphatase and supported membranes by AFM force spectroscopy.
Mammalian alkaline phosphatases (AP) are glycosylphosphatidylinositol (GPI) anchored proteins that are localized on the outer layer of the plasma membrane. The GPI anchors are covalently attached to the C-termini of proteins and consist of a glycan chain bonded to phosphatidylinositol with two acyl chains anchored into the membrane bilayer. Force spectroscopy, based on atomic force microscope (AFM) technology, was used to determine the adhesion of alkaline phosphatase in the absence and presence of anchors. The GPI anchors increase markedly the adhesion frequency (i.e., the protein affinity for the membrane). An adhesion force of 350 +/- 200 pN is measured between GPI-anchored AP (AP(GPI)) and supported phospholipid bilayers of dipalmitoylphosphatidylcholine (DPPC) presenting structural defects (holes). In the absence of defects, the adhesion force (103 +/- 17 pN) and the adhesion frequency are reduced. These results indicate that AP(GPI) poorly spontaneously insert into membranes in vivo and open new perspectives for the characterization of the interactions between GPI proteins and membranes.
Cross
Benjamin
B
Laboratoire de Physique de la Matière Condensée et Nanostructures, Université Claude Bernard Lyon-1 et CNRS, 69622 Villeurbanne, France.
Ronzon
Frédéric
F
Roux
Bernard
B
Rieu
Jean-Paul
JP
D000268:Adhesiveness; D000469:Alkaline Phosphatase / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D007422:Intestines / Q000737:chemistry; D008567:Membranes, Artificial; D018625:Microscopy, Atomic Force / Q000379:methods*; D012680:Sensitivity and Specificity; D013499:Surface Properties
D017261:Glycosylphosphatidylinositols; D008567:Membranes, Artificial; D000469:Alkaline Phosphatase