Man Erythrocyte Membranes The Protein 4.2 And Other Protein Interactions

Protein 4.2 is an important peripheral protein in the erythrocyte membranes, comprising approximately 5% of the total membrane proteins. There are 200,000 copies per cell. The gene that encodes protein 4.2 is located on chromosome 15q. The complete amino acid sequence for human erythrocyte protien 4.2 has been derived from the nucleotide sequence of a full-length 2.35-kilobase (kb) cDNA. Of the 2348 base pairs, 2073 bp encode 691 amino acids representing 76.9 kDa. Protein 4.2 can directly bind to band 3, ankyrin and spectrin. It also associates with protein 4.1 and CD47 which introduces the Rh complex to the membrane skeleton. The N-terminal cytoplasmic domain (cytoplasmic domain of erythrocyte band 3, cdb3) serves as a center of membrane organization in the erythrocytes associates with ankyrin, protein4.1 and protein 4.2, linking cytoskeleton with cell membrane, helping to maintain the mechanical properties and integrity of the erythrocytes.Protein-protein and protein-lipid interactions are thought to play the vital role in maintenance and deformation of the erythrocyte membrane. It has been well demonstrated that the band 3-ankyrin-spectrin bridge and the glycophorin C-protein 4.1-spectrin/actin bridge constitute the two major tethers between the erythrocyte membrane and its spectrin skeleton. Protein 4.2 may promote the linkage between the skeleton network and the plasma membrane. Protein 4.2 becomes more important because the deficiency of protein 4.2 results a borderline variety of hereditary spherocytosis (HS). On the other hand, the structural basis of associations of protein4.2 with band 3, ankyrin, and other membrane proteins in the erythrocyte membranes is still not elucidated in detail. Although there are evidences for interactions of protein 4.2 with cdb3 or ankyrin in the membrane, the exact binding sites and the molecular mechanism of these interactions remain to be characterized and there is no detailed report related to either mapping of ankyrin-binding domain of protein 4.2 or protein 4.2-binding domain on ankyrin till now.We first tried to setup the feasible in vitro research system to study the protein 4.2. We tried to express full length recombinant protein 4.2 in E. coli, but the expression level was poor.According to the result of enzymic digestion of protein 4.2 with staphylococcal V8 protease and CNBr digestion of protein 4.2, we constructed and successfully got eight protein 4.2-derived overlapping recombinant fusion proteins: GST-P4.2(31-200), GST-P4.2 (187-260), GST-P4.2 (155-211), GST-P4.2 (155-260), GST-P4.2 (34-185), GST-P4.2 (100-211), GST-P4.2 (100-260) and Trx-P4.2 (1-300). We also successfully got GST-cdb3 , His6-cdb3 , GST-ankyrinD34 and Trx-ankyrinD34 to study the protein-protein interactions. After optimizing the expression condition, the recombinant proteins were purified by anion exchanger column, affinity column and size exclusive column in series. We verified the purification procedure by SDS-PAGE and Western blot. Their interactions were investigated by using Far-Western blot and Pull-down assay. The results showed that the cdb3-binding site of protein 4.2 is located in the region of residues 200-211 and the ankyrin-binding site is located in the region of residues 187-200 of protein 4.2. Our findings also suggested that the ankyrin D34 domain can interact directly with protein 4.2. The proper tertiary structures of these protein 4.2 fragments are essential for protein 4.2-ankyrin interaction. Meanwhile, ankyrin can enhance the interaction between protein 4.2 and cdb3.The deficiency of protein 4.2 results a borderline variety of hereditary spherocytosis. P 4.2- TOZEUR (GAT>TAT; D175Y) and P4.2- NIPPON (GCT>ACT; A142T) are two kind of known hereditary spherocytosis diseases. Since the residue position 175 is on the linker strand between domains 1 and 2 in the protein 4.2 homology model, replacement of D175 by the larger, neutral tyrosine residue may affect the proper tertiary structures of protein 4.2. In order to study the effects of 3 protein-4.2 point mutations associated with HS, we have cloned four recombinant fusion proteins: GST-P4.2 (31-200) D175Y, GST-P4.2 (31-200) D175Y, GST-P4.2 (100-260) D175Y, GST-P4.2 (31-200) A142T with pointmutation, to do prokaryotic expression, and tested their interactions with ankyrin by Far-Western blot and Pull-down assay, but there were no evidences to show these mutated proteins can bind to ankyrin except GST-P4.2 (31-200) A142T.In order to reveal the role how D175Y proteins do no bind to ankyrin, we have constructed three point-mutated proteins: GST-P4.2 (31-200) D175K, GST-P4.2 (31-200) D175F and GST-P4.2 (31-200) D175A. Their interactions with ankyrin were studied by using Far-Western blot and Pull-down assay. The results showed that GST-P4.2 (31-200) D175K could interact with ankyrin while other two could not.The results showed that replacement of D175 by tyrosine may break the salt bridge and disrupt the domain l/domain2 interface of protein 4.2, leading its not binding to ankyrin.