Purification and characterization of the human erythrocyte band 3 protein C-terminal domain.

To clarify the function of the hydrophilic carboxyl-terminal tail of human erythrocyte membrane band 3 protein (HEM-B3), we purified two peptides, C1 (Ala893-Val911) and KS4 (Gly647-Arg656), from human erythrocyte band 3 protein preparations. Purified C1 peptides at concentrations from 5 to 80 microM were incubated with fresh human erythrocyte white ghosts. The C1 peptide demonstrated a novel protease activity, which cleaved glycophorin A (GPA) at Leu118-Ser119 in a dose-dependent manner. This activity was eliminated by trypsin. In a control experiment, the KS4 peptide did not cleave GPA under the same conditions. To help substantiate that the band 3 C-terminal tail peptide (C1) alone possesses the protease activity, two experiments were performed. First, the plasmids pGBKT(7)-GPA-Ct and pGADT(7)-AE1-Ct were cotransformed into the yeast strain AH109. The pGBKT(7)-GPA-Ct plasmid contains the cDNA of the 33 amino acid residue section of GPA (Tyr93-Asn125) fused with the pGBKT(7) vector. The plasmid pGADT(7)-AE1-Ct contains the cDNA of the C-terminal 33 amino acid residues of HEM-B3 fused with the GAL4 DNA-binding domain in the pGADT(7) vector. The results of the cotransformation experiment indicated that the C-terminal 33 amino acid residues of HEM-B3 interacted directly with the GPA C-terminal segment defined above. Second, we used a mammalian two-hybrid analysis to confirm the interaction relationship between the band 3 C-terminal segment and the GPA C-terminus. The C-terminus of GPA and the C-terminal 33 amino acid residues of HEM-B3 were subcloned into the DNA-binding domain and transcription activation domain vectors of the two-hybrid system, respectively. They were then cotransfected along with a chloramphenicol acetyltransferse (CAT) reporter vector into HeLa cells. The CAT activity measured in this experiment also indicated that there was interaction between the C-terminal 33 amino acid residues of HEM-B3 and the C-terminus of GPA.