Molecular characterization of mouse monoclonal antibody BIII.136 and the epitope recognized by the antibody in human band 3 protein.
The monoclonal antibody BIII.136, which recognizes the cytoplasmic part of the band 3 protein from human erythrocytes, also detects products of proteolytic degradation of that protein caused by endogeneous proteases in erythrocytes. Now we extend and confirm these observations by finding that in very young erythrocytes from patients with hemolytic anemias the band 3 protein is almost intact, which suggests that proteolytic degradation of that protein proceeds in vivo during the life span f the erythrocyte. Interesting properties and applicability of this antibody for following the band 3 degradation in vivo and for detection of the band 3 variant forms have prompted us to characterize its primary structure and the epitope recognized in band 3. A set of solid phase-synthesized peptides allowed us to establish that MAb BIII.136 is directed against sequence EDPDIP, which corresponds to amino acid residues 22-27 in band 3 protein. Replacement analysis revealed that only E22 and P24 can be replaced by several other amino acids without a significant loss of reactivity, while the remaining four amino acids seem to be an essential part of the epitope. No reactivity of the antibody with band 3 from several other species was found. Analysis of the heavy and light chain variable region cDNAs revealed that the VH is encoded by a member of VH8(VH3609) family, while the VL is encoded by a member of the Vk12/13 family.
Czerwiński
M
M
Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw.
Usnarska-Zubkiewicz
L
L
D000595:Amino Acid Sequence; D000740:Anemia / Q000097:blood / Q000276:immunology; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000276:immunology* / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001483:Base Sequence; D001666:Binding Sites, Antibody; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry*; D004912:Erythrocytes / Q000276:immunology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data
D001457:Anion Exchange Protein 1, Erythrocyte; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007135:Immunoglobulin Variable Region