The SH3 Domain of Alpha Spectrin Binds to Galectin-1 during Erythroid Differentiation.
Authors: Lima Paulo R.M. , Baratti Mariana O. , Basseres Daniela S. , Duarte Adriana S.S. , Costa Fernando F. , Saad Sara T.O. 
Published: April 2020

Abstract
A major function of the spectrin skeleton in erythrocytes is to provide mechanical support for the membrane bilayer and allow survival of these cells in the circulation. An interesting feature of spectrin is that the alpha subunit contains a highly conserved Src homology 3 domain (SH3) of unknown function. The SH3 domains are small protein-protein interaction domains that mediate a range of important biological processes and are considered valuable targets for the development of therapeutic agents. A possible direct role of spectrin in signal transduction, recruiting or activating signaling molecules through the SH3 domain, has been recently explored. It seems that spectrin and its SH3 domain promote Rac activation in the specialized integrin cluster which initiate cell adhesion and spreading. In the present study, we used the yeast two-hybrid system to screen a human bone marrow cDNA library, looking for a protein that interacts with a 62 aa sequence containing the SH3 domain of spectrin, which comprises Ala977 to Glu1038 of alpha-spectrin protein (SPTA1). We screened 5.9 x 105 clones from a human bone marrow cDNA library. Thirteen positive clones were obtained, of which two contained the region K51-K123 of Niemann Pick C2 protein (NP-C2), one contained the region A2-M121 of the galectin 1 (LGALS1) and one contained the region P4-F128 of interleukin 2 receptor gamma (IL2RG). We considered the possible interaction between spectrin and galectin-1 (GAL1), a beta-galactoside-binding lectin involved in cell cycle progression. Galectin-1 over expression is associated with neoplastic transformation and loss of differentiation. In order to verify the physical association between galectin-1 and erythroid spectrin we differentiated erythroid progenitor cells obtained from peripheral blood mononuclear cells to erythroblasts stages, subsequent to the proerythroblast stage, using a two phase liquid culture system. Cells were collected at day zero, 7 and 13 of the second phase. FACS analysis using glicophorin A and transferrin receptor antibodies confirmed the erythroid differentiation at days 7 and 13. Cellular extracts were immunoprecipitated with a goat polyclonal anti-galectin-1 antibody (Santa Cruz) and the proteins were submitted to 6% SDS-PAGE and transferred to a nitrocellulose membrane which was immunobloted with a rabbit polyclonal anti-spectrin alpha 1 antibody (Santa Cruz). At the 13th day of differentiation, the immunoblotting showed a 240 kDa band, compatible to the alpha spectrin molecular weight, suggesting the physical association between erythroid alpha spectrin and galectin-1. Corroborating our data, Marion et al (Mol Biochem Parasitol. 2004, 135(1):31–8) recently described a protein from the spectrin family co-localizing with the Gal-GalNAc lectin in Entamoeba histolytica and confirmed this interaction by in vitro assay. The acquisition of an erythroid phenotype is associated with externalization of galectin-1. Spectrin may have a role in initiating the externalization of galectin-1, an event related to late stages of erythroid differentiation and this association might involve conformational changes mediated by SH3 domain of erythrocyte alpha spectrin. The autocrine binding of galectin-1 to cell surface ligands of nonadherent cells such as K562 suggest that galectin-1 is implicated in signal transduction rather than in cell-cell or cell-matrix interaction. In conclusion, this is the first report identifying the interaction between galectin-1 and the SH3 domain of alfa-spectrin in erythroid cell.