Purification and Characterization of the Human Erythrocyte Band 3 Protein C-Terminal Domain

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• 作者：Guohui Fu ; Tianying Wang ; Baofeng Yang ; Fengxiang Lv ; Congning Shi ; Xiaoshu Jiang ; Lifeng Tian ; Weihan Yu ; and Naotaka Hamasaki
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：February 17, 2004
• 年：2004
• 卷：43
• 期：6
• 页码：1633 - 1638
• 全文大小：109K
• 年卷期：v.43,no.6(February 17, 2004)
• ISSN：1520-4995

文摘

To clarify the function of the hydrophilic carboxyl-terminal tail of human erythrocyte membraneband 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 80M were incubated with fresh human erythrocyte white ghosts. The C1 peptide demonstrated a novelprotease 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 GPAunder the same conditions. To help substantiate that the band 3 C-terminal tail peptide (C1) alone possessesthe protease activity, two experiments were performed. First, the plasmids pGBKT₇-GPA-Ct and pGADT₇-AE1-Ct were cotransformed into the yeast strain AH109. The pGBKT₇-GPA-Ct plasmid contains thecDNA of the 33 amino acid residue section of GPA (Tyr93-Asn125) fused with the pGBKT₇ vector.The plasmid pGADT₇-AE1-Ct contains the cDNA of the C-terminal 33 amino acid residues of HEM-B3fused with the GAL4 DNA-binding domain in the pGADT₇ vector. The results of the cotransformationexperiment indicated that the C-terminal 33 amino acid residues of HEM-B3 interacted directly with theGPA C-terminal segment defined above. Second, we used a mammalian two-hybrid analysis to confirmthe interaction relationship between the band 3 C-terminal segment and the GPA C-terminus. TheC-terminus of GPA and the C-terminal 33 amino acid residues of HEM-B3 were subcloned into theDNA-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 intoHeLa cells. The CAT activity measured in this experiment also indicated that there was interaction betweenthe C-terminal 33 amino acid residues of HEM-B3 and the C-terminus of GPA.