Specificity of human IgM M-proteins that bind to myelin-associated glycoprotein: peptide mapping, deglycosylation, and competitive binding studies.
Ten patients with neuropathy and IgM M-proteins that bind to the myelin-associated glycoprotein (MAG) were studied to determine whether the M-proteins bind to common regions of MAG and whether the reactive determinants contain carbohydrate residues. The M-protein of one patient was biotinylated, and binding to human MAG was quantitated by enzyme-linked immunosorbent assay (ELISA) by using avidin-biotin-peroxidase complexes. Serum from the same patient and nine others, but not from controls, competed with the biotinylated M-protein for binding to human MAG. Bovine MAG was digested with staph protease or cleaved with cyanogen bromide, and the resultant fragments were separated by electrophoresis and were transferred onto nitrocellulose sheets. Serum from all patients immunostained the peptide fragments identically. Bovine MAG was deglycosylated by trifluoromethanesulfonic acid, and binding of the M-proteins to MAG and to deglycosylated MAG was tested by immunoblotting. None of the patient's M-proteins bound to deglycosylated MAG. Deglycosylated MAG was visualized by using a mouse monoclonal antibody, GEN-S3, directed at the polypeptide core of MAG. The effectiveness of deglycosylation was ascertained by electrophoresis and by binding of biotinylated concanavalin A. These data and the observed identical species specificity of the M-proteins suggest that the respective anti-MAG M-proteins all bind to the same region in MAG and that the reactive determinants may contain carbohydrate moieties.
D000818:Animals; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D001798:Blood Proteins / Q000378:metabolism*; D002417:Cattle; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007124:Immunoenzyme Techniques; D007075:Immunoglobulin M / Q000378:metabolism*; D007136:Immunoglobulins; D008698:Mesylates / Q000494:pharmacology; D009185:Myelin Proteins / Q000378:metabolism*; D018992:Myelin-Associated Glycoprotein; D010455:Peptides / Q000302:isolation & purification
D001798:Blood Proteins; D007075:Immunoglobulin M; D007136:Immunoglobulins; C009823:M-proteins (Myeloma); D008698:Mesylates; D009185:Myelin Proteins; D018992:Myelin-Associated Glycoprotein; D010455:Peptides; D003101:Collodion; C012077:trifluoromethanesulfonic acid