Degradation of the human erythrocyte membrane band 3 studied with the monoclonal antibody directed against an epitope on the cytoplasmic fragment of band 3

The mouse hybridoma monoclonal antibody BIII.136 of the IgG2a class is specific for human erythrocyte band-3 protein. It was shown by means of immunoblotting and immunoprecipitation assays that the antibody recognized an epitope located in the cytoplasmic pole of the band-3 molecule within approximately 20 kDa from the N-terminal end. The N-terminal fragments of band-3 protein, migrating in SDS/polyacrylamide gel electrophoresis in the 60-kDa, 40-kDa and 20-kDa regions, were detected with the antibody in untreated red-cell membranes as products of autolysis of band-3 protein. A correlation was found between the amount of these fragments and erythrocyte age, which suggests that partial degradation of band 3 proceeds in vivo during senescence of erythrocytes. The further degradation of band-3 protein iiz vitro was not observed in intact erythrocytes stored at 4"C, but progressed distinctly after hemolysis of red cells, during washing and storing the membranes. The band-3 protein is the most abundant integral protein of human erythrocyte membranes [I] and functions as an anion transporter (cf. [2-41). Extensive studies on the structure, disposition in the membrane and biological function of band 3 are reviewed in several articles [2 - 81. Band 3 is a 90 100-kDa protein, its microheterogeneity is ascribed mainly to the structural heterogeneity of the carbohydrate moiety. The C-terminal portion of the molecule spans the membrane several times, is responsible for anion transport and carries one large N-linked oligosaccharide chain of the polylactosamine type, located outside the red cell. The N-terminal portion, comprising almost half of the polypeptide chain of band 3, is located inside the red cell and interacts with cytoskeletal and cytoplasmic proteins, such as ankyrin, bands 4.1 and 4.2, hemoglobin and some glycolytic enzymes (cf. [5, 81). Band 3 is a highly hydrophobic protein, it exists in erythrocytes as a dimer and tetramer and has a strong tendency to aggregate Proteins similar to human erythrocyte band 3 are present in animal erythrocytes and, in smaller amount, in nonerythroid cells (cf. [8]). Recently the complete amino acid sequences of band 3 from mouse erythrocytes and band-3like protein from the human leukemic cell line K562 have been deduced from the nucleotide sequences of the respective cDNAs [9, lo]. Comparison of these predicted sequences and a partial amino acid sequence of human erythrocyte band 3