Alteration Young's moduli by protein 4.1 phosphorylation play a potential role in the deformability development of vertebrate erythrocytes

The mechanical properties of vertebrate erythrocytes depend on their cytoskeletal protein networks. Membrane skeleton proteins spectrin and protein 4.1 (4.1R) cross-link with actin to maintain membrane stability under mechanical stress. Phosphorylation of 4.1R alters the affinity of 4.1R for spectrin-actin binding and this modulates the mechanical properties of human erythrocytes. In this study, phorbol 12-myristate-13-acetate (PMA)-induced phosphorylation of 4.1R was tested, erythrocyte deformability was determined and the erythrocyte elastic modulus was detected in human, chick, frog and fish. Furthermore, amino acid sequences of the functionally important domains of 4.1R were analyzed. Results showed that PMA-induced phosphorylation of 4.1R decreased erythrocyte deformability and this property was stable after 1 h. The values of Young's modulus alteration gradually decreased from human to fish (0.388±0.035 kPa, 0.219±0.022 kPa, 0.191±0.036 kPa and 0.141±0.007 kPa). Ser-312 and Ser-331 are located within the consensus sequence recognized by protein kinase C (PKC); however, Ser-331 in zebrafish was replaced by Ala-331. The sequence of the 8 aa motif from vertebrate 4.1R showed only one amino acid mutation in frog and numerous substitutions in fish. Analyses of Young's modulus suggested that the interaction between 4.1R with the spectrin-actin binding domain may have a special relationship with the development of erythrocyte deformability. In addition, amino acid mutations in 4.1R further supported this relationship. Thus, we hypothesize that alteration of membrane skeleton protein binding affinity may play a potential role in the development of erythrocyte deformability, and alteration of Young's modulus values may provide a method for determining the deformability development of vertebrate erythrocytes.