Effect of gangliosides on the distribution of a glycosylphosphatidylinositol-anchored protein in plasma membrane from Chinese hamster ovary-K1 cells.
Glycosylphosphatidylinositol (GPI)-anchored proteins are clustered mainly in sphingolipid-cholesterol microdomains of the plasma membrane. The distribution of GPI-anchored fusion yellow fluorescent protein (GPI-YFP) in the plasma membrane of Chinese hamster ovary (CHO)-K1 cells with different glycolipid compositions was investigated. Cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity or cell lines expressing different gangliosides caused by stable transfection of appropriate ganglioside glycosyltransferases or exposed to exogenous GM1 were transfected with GPI-YFP cDNA. The distribution of GPI-YFP fusion protein expressed at the plasma membrane was studied using the membrane-impermeable cross-linking agent bis(sulfosuccinimidyl)suberate. Results indicate that GPI-YFP forms clusters at the surface of cells expressing GM3, or cells depleted of glycolipids, or transfected cells expressing mainly GD3 and GT3, or GM1 and GD1a, or mostly GM2, or highly expressing GM1. However, no significant changes in membrane microdomains of GPI-YFP were detected in the different glycolipid environments provided by the membranes of the cell lines under study. On the other hand, wild type CHO-K1 cells exposed to 100 microm GM1 before cross-linking with bis(sulfosuccinimidyl)suberate showed a dramatic reduction in the amount of GPI-YFP clusters. These findings clearly indicate that manipulating the glycolipid content of the cellular membrane, just by changing the ganglioside biosynthetic activity of the cell, did not significantly affect the association of GPI-YFP on the cell surface of CHO-K1 cells. The effect of exogenous GM1 gangliosides on GPI-YFP plasma membrane distribution might be a consequence of the ganglioside level reached in plasma membrane and/or the effect of particular ganglioside species (micelles) that lead to membrane architecture and/or dynamic modifications.
Crespo
Pilar Maria
PM
Centro de Investigaciones en Química Biológica de Córdoba, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba 5000, Argentina.
Zurita
Adolfo Ramón
AR
Daniotti
Jose Luis
JL
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D005677:G(M1) Ganglioside / Q000378:metabolism / Q000494:pharmacology*; D005964:Glucosyltransferases / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013388:Succinimides / Q000494:pharmacology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D016895:Culture Media, Serum-Free; C028391:G protein, vesicular stomatitis virus; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D013388:Succinimides; D014759:Viral Envelope Proteins; C054562:yellow fluorescent protein, Bacteria; D005677:G(M1) Ganglioside; C035760:bis(sulfosuccinimidyl)suberate; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase