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. Th e experiments were performed with erythrocyte membrane proteins, in wh ich glycophorins are the major poly-O-glycosylated components, and wit h lysates of human colon cancer cells CX-1.1. Lectins and monoclonal a ntibodies against peptidic, glycopeptidic, and carbohydrate epitopes w ere used to examine the effect of degradation. Experiments with erythr ocyte 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 unde tectable, while N-glycans and peptidic epitopes of proteins were detec ted with unchanged or even increased intensity compared to untreated b lots. The method was used to show that most protein-linked sialyl-Le(a ) 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 e pitopes of glycophorin A (GPA). Most of these antibodies recognize gly copeptidic epitopes dependent on O-glycosylation and, therefore, they did not detect GPA on NaOH-treated blots. Some less frequent anti-M an tibodies cross-reacting with the rare GPA variant of M-g type are spec ific for a peptidic epitope which is unrelated to the MN blood group-s pecific amino acid sequence in unglycosylated peptides, but is recogni zed 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. (C) 1997 Academic Press.