Recombinant forms of glycophorin C as a tool for characterization of epitopes for new murine monoclonal antibodies with anti-glycophorin C specificity.
Glycophorin C (GPC) and glycophorin D (GPD) are minor but important components of human RBC membranes. They carry the high-frequency antigens Ge2, Ge3 and Ge4 of the Gerbich blood group system. The epitopes for five new monoclonal antibodies (MoAbs) with anti-GPC specificity were characterized. Two antibodies (4G11 and 5B11) reacted with glycosylated N-terminal epitopes, and three reacted with internal epitopes of GPC. Pepscan analysis showed that the MoAb RB11 required for binding the EPDP sequence, occurring twice in GPC polypeptide chain. The MoAb 7F11 recognized the sequence 13PLSLEPDP20, and the MoAb RB8 did not react with synthetic peptides. Further characterization of the internal epitopes was performed in fluorescence-activated cell sorter (FACS) with the use of recombinant GPC and its variant forms transiently expressed on COS-7 cells. The results indicated that the MoAb RB11 recognized distinctly its target sequence EPDP only in a normal GPC molecule. The reactivity of the MoAb 7F11 with the PLSLEPDP sequence was confirmed and found to be enhanced by the O-glycan at the Ser15 residue. The MoAb RB8 recognized the glycopeptidic epitope in proximity to the Ser15 residue, requiring the presence of O-glycan. The combination of immunochemical techniques with the use of the recombinant forms of GPC has made it possible to define the role of sugar chains in the recognition of peptidic epitopes in glycosylated antigen and sheds new light on the Gerbich system antigens.
Jaskiewicz
E
E
Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. jaskiew@immuno.iitd.pan.wroc.pl
Czerwinski
M
M
Uchikawa
M
M
Murata
S
S
Miyazaki
T
T
Ikeda
H
H
Lisowska
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D018604:Epitope Mapping; D005434:Flow Cytometry; D006021:Glycophorins / Q000276:immunology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000276:immunology; D017384:Sequence Deletion
D000911:Antibodies, Monoclonal; D006021:Glycophorins; D011994:Recombinant Proteins