Erythroid Alpha Research Articles

The SH3 Domain of Alpha Spectrin Binds to Galectin-1 during Erythroid Differentiation.

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.

Constitutive and inducible expression of megakaryocyte-specific genes in Friend erythroleukaemia cells.

Friend murine erythroleukaemia cells (MELC) were analysed by semiquantitative RT-PCR for the constitutive and inducible expression of megakaryocyte-specific genes. Uninduced MELC expressed detectable levels of mRNAs for acethylcholinesterase (AChE), platelet factor-4 (PF4), glycoprotein IIb (GPIIb) and von Willebrand factor (VWF), whereas the erythroid alpha- and beta-globin genes were not transcribed appreciably. However, MELC exposed to 5 mM hexamethylene bisacetamide (HMBA) or 1.5% dimethyl sulphoxide (DMSO) seemed to be channelled towards a mixed erythroid/megakaryocytic phenotype characterized by unaltered levels of VWF mRNA, increased levels of AChE, GPIIb and PF4 mRNA. and simultaneous induction of the globin genes. Megakaryocyte-related genes were expressed. in the absence of globin gene transcription, by MELC treated with either phorbol-12-myristate acetate (PMA; 100 ng/ml) or colcemid (40 nM), an antimicrotubule agent capable of promoting polyploidization in this model. Moreover, PMA and colcemid induced also de novo expression of the thrombopoietin receptor c-mpl. PMA and colcemid did not affect high basal c-myb mRNA levels which, in turn, were down-regulated upon HMBA or DMSO induction. Additionally, both uninduced and induced MELC exhibited significant levels of Epo-R and IL-3R mRNAs, whereas no expression of granulocyte/macrophage-related genes was detected. Megakaryocyte gene expression of MELC was also compared to that of other haemopoietic cell populations from normal mice and mice infected with the anaemic strain of the Friend virus. According to our results, MELC should be seen as an unique erythro-megakaryocytic model of differentiation, potentially useful for studying molecular events governing lineage commitment as well as some steps of megakaryocytopoiesis.

Human cardiac and skeletal muscle spectrins: differential expression and localization.

We describe multiple human cardiac and skeletal muscle spectrin isoforms. Cardiac muscle expresses five erythroid alpha,beta spectrin-reactive isoforms with estimated MR's of 280, 274, 270, 255, and 246 kD, respectively. At least one nonerythroid alpha-spectrin of MR 284 kD is expressed in heart. While skeletal muscle shares the 280, 270, and 246 kD erythroid spectrins, it expresses an immunologically distinct 284 kD nonerythroid alpha-spectrin isoform. The 255 kD erythroid beta-spectrin isoform is specific for cardiac tissue. By immunocytochemistry, both erythroid beta- and nonerythroid alpha-spectrins are localized to costameres, the plasma membrane, and the neuromuscular junctional region.

Characterization of the Calmodulin-binding Site of Nonerythroid α-Spectrin: Recombinant protein and model peptide studies

An important function of the mammalian nonerythroid alpha-spectrin chain (alpha-fodrin) that distinguishes it from the closely related erythroid isoform is its ability to bind calmodulin. By analysis of a series of deleted recombinant spectrin fusion proteins, we have identified a region in the nonerythroid alpha chain involved in calcium-dependent binding of calmodulin. The region is distinctive in that the sequence is absent from the homologous domain of the erythroid alpha chain and diverges from the normal internal repeat structure observed throughout other spectrins. In order to determine limits of this functional site, a synthetic peptide as small as 24 residues was shown to compete with either recombinant or brain alpha-spectrin in binding to calmodulin. The active peptide, which was derived from a segment between repeats 11 and 12, was composed of the following sequence: Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala-Thr-Phe-Asn - Ser-Ile-Lys-Glu. Comparison of this sequence with functional sites in other diverse calcium-dependent calmodulin-binding proteins has revealed a structural motif common to all of these proteins, namely clusters of hydrophobic residues interspersed with basic residues. When folded into alpha-helical conformations, these binding sites are predicted to form amphipathic structures.