Evaluation by dot-immunoassay of the differential distribution of cell surface and intracellular proteins in glycosylphosphatidylinositol-rich plasma membrane domains.
The dot-immunoassay has been adapted for rapid detection and partial characterization of glycosylphosphatidylinositol (GPI)-linked, transmembrane, and intracellular proteins in Triton X-100 (TX-100) extracts of lymphoma cells and intestinal tissue. The GPI-anchored proteins tend to concentrate into specialized plasma membrane domains enriched in glycosphingolipids. The dot-immunoassay has been successfully used to demonstrate the differential distribution of GPI-linked and transmembrane surface glycoproteins of T lymphocytes in sucrose density gradient fractions of TX-100 lysate. The type II transmembrane protein CD26 and the intracellular tyrosine kinase p56lck partially cofractionated with GPI-linked glycoproteins, and the extent to which they partition into GPI-rich plasma membrane domains could be evaluated. Preferential association of the acidic glycosphingolipid GM1 with these domains could be demonstrated by cholera toxin binding directly to the dot-blotted sucrose density gradient fractions. Treatment of whole cell TX-100 lysates or sucrose gradient fractions dotted onto nitrocellulose filter strips with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) proved to be an efficient method to assay for the presence of a GPI-anchor in Thy-1 and Ly6 surface glycoproteins. We have used three criteria, namely flotation to light density fractions in sucrose gradients, colocalization with GM1, and sensitivity to PI-PLC cleavage, to assess the presence of a GPI modification in a putative GPI-linked protein in intestinal tissue extract. It is envisaged that the techniques described in this report would find a wider application to rapidly assess the contents of GPI-rich plasma membrane domains in different cells and tissues.
Ilangumaran
S
S
Department of Pathology, Centre Médical Universitaire, Geneva, Switzerland.
Arni
S
S
Chicheportiche
Y
Y
Briol
A
A
Hoessli
D C
DC
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007118:Immunoassay; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D017830:Octoxynol; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D017830:Octoxynol; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase