title: The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis.
abstract: Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation.
authors.affiliation: School of Biochemistry, Medical Sciences Building, University Walk, Bristol, UK Bristol Institute of Transfusion Sciences, NHSBT Filton, Bristol, UK t.satchwell@bristol.ac.uk ash.m.toye@bristol.ac.uk.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Satchwell
authors.affiliation: School of Biochemistry, Medical Sciences Building, University Walk, Bristol, UK Bristol Institute of Transfusion Sciences, NHSBT Filton, Bristol, UK.
authors.forename: Bethan R
authors.initials: BR
authors.lastname: Hawley
authors.affiliation: School of Biochemistry, Medical Sciences Building, University Walk, Bristol, UK.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Bell
authors.affiliation: Servico de Hematologia Clinica, Centro Hospitalar e Universitario de Coimbra, Portugal.
authors.forename: M Leticia
authors.initials: ML
authors.lastname: Ribeiro
authors.affiliation: School of Biochemistry, Medical Sciences Building, University Walk, Bristol, UK Bristol Institute of Transfusion Sciences, NHSBT Filton, Bristol, UK t.satchwell@bristol.ac.uk ash.m.toye@bristol.ac.uk.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Toye
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017487:Ankyrins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D016022:Case-Control Studies; D002454:Cell Differentiation; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004920:Erythropoiesis / Q000502:physiology*; D005434:Flow Cytometry; D006720:Homozygote; D006801:Humans; D046912:Multiprotein Complexes / Q000378:metabolism*; D010641:Phenotype; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D012156:Reticulocytes / Q000166:cytology / Q000378:metabolism; D013103:Spherocytosis, Hereditary / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D046912:Multiprotein Complexes; D034741:RNA, Small Interfering