The murine monoclonal antibody NaM26-4C6 identifies a common structure on band 3 and glycophorin C

The murine monoclonal antibody NaM26-4C6 (IgM class), obtained from the spl enocytes of a BALB/c mouse immunized with human umbilical cord red blood ce lls, was characterized by agglutination test and immunoblotting analysis. T he structure of the NaM26-4C6 epitope was further elucidated by using a ser ies of peptides synthesized on pins. The antibody agglutinated untreated an d chymotrypsin-treated but not trypsin- or neuraminidase-treated human eryt hrocytes. Agglutination-inhibition test demonstrated that the antibody reco gnizes an epitope located on the N-terminal trypsin-sensitive portion of gl ycophorin C. The antibody bound on immunoblots to glycophorin C, and also t o the band 3 protein and its 69-kDa N-terminal fragment but did not bind to desialylated and de-O-glycosylated glycophorin C. Peptide mapping allowed localization of the binding site on the 23-kDa N-terminal intracellular pep tide of band 3, The antibody binds to the amino-acid sequences (EDPDIP27)-E -22 of band 3 protein and (15)SLEPDPGM(22) of glycophorin C, and residues D and P were found to be essential. The new epitope identified by NaM26-4C6 corresponds to a linear amino acid sequence located on the N-terminal intra cellular portion of band 3 and to a more complex structure involving oligos accharide chains on the N-terminal extracellular domain of GPC.