Effects of gangliosides on the distribution of to GPI-anchored protein in plasm membrane

DANIOTTI, J.L.; CRESPO P; ZURITA A.R

Palm Beach, FL, USA

Congreso; 33rd Annual Congress of the American Society of Neurochemistry 2002; 2002

American Society of Neurochemistry

GPI-anchored proteins are mainly clustered in sphingolipid-cholesterol microdomains of the plasma membrane (PM), which are characterized by a high insolubility in Triton X-100. We investigate here the distribution of a GPI-anchored chimera, which contains an ER-import signal, and the total sequence of the yellow fluorescence protein fused to a GPI-attachment signal (GPI-YFP) in CHO-K1 cells with different glycolipid compositions. Cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity, or cell lines expressing different gangliosides due to stable transfection of appropriate ganglioside glycosyltransferases, or treated with exogenous GM1 were used. The association of GPI-YFP chimera on the cell surface was investigated by using the membrane-impermeable cross-linking agent bis(sulphosuccinimidyl)suberate (BS3). Parental cell expressing GM3, or cells depleted of glycolipids, or transfected cells expressing mostly GM1 and GD1a or GD3 and GT3 subjected to chemical cross-linking with BS3 showed a major ~80 kDa band (dimer) and a band of high molecular weight (>400 kDa) detected by Western blots. However, minor changes in the relative proportion between these two bands were detected in the different cell clones. On the other hand, parental cells loaded with varying concentration (100-300mM) of GM1 for 1 h before cross-linking with BS3 showed a dramatic reduction in the efficiency of cross-linking. Results indicate that loading with exogenous GM1 results in formation of particular species that alter the GPI-YFP membrane distribution. These species would not form when the composition of gangliosides is changed keeping normal the process of synthesis and insertion into membranes.