Mapping of a spectrin-binding domain of human erythrocyte membrane protein 4.2.
Protein 4.2 is a major component of the red blood cell membrane skeleton. Deficiency of protein 4.2 is linked with a variety of hereditary haemolytic anaemias. However, the interactions of protein 4.2 with other proteins of the erythrocyte membrane remain poorly understood. The major membrane-binding site for protein 4.2 resides on the cytoplasmic domain of band 3. Protein 4.2 interacts directly with spectrin in solution, suggesting that it stabilizes interactions between the membrane skeleton and the erythrocyte membrane. A 30 kDa polypeptide, with its N-terminus corresponding to amino acid residue 269, derived by partial proteolysis of protein 4.2, was found to interact with biotinylated spectrin in gel renaturation assays. A series of overlapping glutathione S-transferase fusion peptides were constructed, and an alpha-helical domain encompassing residues 470-492 was found to be instrumental in mediating protein 4.2-spectrin interactions. Direct binding of a synthetic peptide, with the sequence corresponding to residues 470-492, to spectrin and the ability of the peptide to inhibit spectrin binding of protein 4.2 confirmed that these residues are crucial in mediating protein 4.2-spectrin interactions.
Mandal
Debabrata
D
Department of Chemistry, Bose Institute, 93/1 Acharya, Prafulla Chandra Road, Kolkata 700 009, India.
Moitra
Prasun K
PK
Basu
Joyoti
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003598:Cytoskeletal Proteins; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013049:Spectrin / Q000378:metabolism*
D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C044791:erythrocyte membrane band 4.2 protein; D013049:Spectrin; D005979:Glutathione Peroxidase