beta-Elimination of O-glycans from glycoproteins transferred to immobilon P membranes: method and some applications.
Selective beta-elimination of O-glycans from glycoproteins transferred from electrophoretic gels onto Immobilon P membranes is described. The experiments were performed with erythrocyte membrane proteins, in which glycophorins are the major poly-O-glycosylated components, and with lysates of human colon cancer cells CX-1.1. Lectins and monoclonal antibodies against peptidic, glycopeptidic, and carbohydrate epitopes were used to examine the effect of degradation. Experiments with erythrocyte membrane proteins showed that after heating the blots in 0.055 M NaOH for 16 h at 40 degrees C the O-glycans of glycophorins were undetectable, while N-glycans and peptidic epitopes of proteins were detected with unchanged or even increased intensity compared to untreated blots. The method was used to show that most protein-linked sialyl-Lea epitopes present on CX-1.1 cancer cells are located on O-glycosidic chains. Moreover, beta-elimination on the blots allows examination of the dependence of peptidic epitopes on O-glycosylation. This was shown using monoclonal antibodies specific for blood group M- or N-related epitopes of glycophorin A (GPA). Most of these antibodies recognize glycopeptidic epitopes dependent on O-glycosylation and, therefore, they did not detect GPA on NaOH-treated blots. Some less frequent anti-M antibodies cross-reacting with the rare GPA variant of Mg type are specific for a peptidic epitope which is unrelated to the MN blood group-specific amino acid sequence in unglycosylated peptides, but is recognized in GPA-M only in the glycosylated antigen. These antibodies, which showed specificity for GPA-M on untreated blots, detected GPA-M, GPA-N, and glycophorin B on NaOH-treated blots.
Duk
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M
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolf Weigl St. 12, Wroclaw, 53-114, Poland.
Ugorski
M
M
Lisowska
E
E
D000911:Antibodies, Monoclonal; D003110:Colonic Neoplasms; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004910:Erythrocyte Membrane / Q000737:chemistry; D006021:Glycophorins / Q000737:chemistry* / Q000302:isolation & purification; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D037102:Lectins; D008562:Membrane Glycoproteins / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D008567:Membranes, Artificial; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011145:Polyvinyls; D012794:Sialic Acids; D014407:Tumor Cells, Cultured; D005465:Fluorocarbon Polymers
D000911:Antibodies, Monoclonal; D006021:Glycophorins; D006023:Glycoproteins; D037102:Lectins; D008562:Membrane Glycoproteins; D008567:Membranes, Artificial; D011134:Polysaccharides; D011145:Polyvinyls; D012794:Sialic Acids; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers