title: Severe Ankyrin-R deficiency results in impaired surface retention and lysosomal degradation of RhAG in human erythroblasts.
abstract: Ankyrin-R provides a key link between band 3 and the spectrin cytoskeleton that helps to maintain the highly specialized erythrocyte biconcave shape. Ankyrin deficiency results in fragile spherocytic erythrocytes with reduced band 3 and protein 4.2 expression. We use in vitro differentiation of erythroblasts transduced with shRNAs targeting ANK1 to generate erythroblasts and reticulocytes with a novel ankyrin-R 'near null' human phenotype with less than 5% of normal ankyrin expression. Using this model, we demonstrate that absence of ankyrin negatively impacts the reticulocyte expression of a variety of proteins, including band 3, glycophorin A, spectrin, adducin and, more strikingly, protein 4.2, CD44, CD47 and Rh/RhAG. Loss of band 3, which fails to form tetrameric complexes in the absence of ankyrin, alongside GPA, occurs due to reduced retention within the reticulocyte membrane during erythroblast enucleation. However, loss of RhAG is temporally and mechanistically distinct, occurring predominantly as a result of instability at the plasma membrane and lysosomal degradation prior to enucleation. Loss of Rh/RhAG was identified as common to erythrocytes with naturally occurring ankyrin deficiency and demonstrated to occur prior to enucleation in cultures of erythroblasts from a hereditary spherocytosis patient with severe ankyrin deficiency but not in those exhibiting milder reductions in expression. The identification of prominently reduced surface expression of Rh/RhAG in combination with direct evaluation of ankyrin expression using flow cytometry provides an efficient and rapid approach for the categorization of hereditary spherocytosis arising from ankyrin deficiency.
authors.affiliation: School of Biochemistry, University of Bristol, UK National Institute for Health Research Blood and Transplant Research Unit (NIHR BTRU) in Red Blood Cell Products, University of Bristol, UK ash.m.toye@bristol.ac.uk t.satchwell@bristol.ac.uk.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Satchwell
authors.affiliation: School of Biochemistry, University of Bristol, UK.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Bell
authors.affiliation: School of Biochemistry, University of Bristol, UK National Institute for Health Research Blood and Transplant Research Unit (NIHR BTRU) in Red Blood Cell Products, University of Bristol, UK.
authors.forename: Bethan R
authors.initials: BR
authors.lastname: Hawley
authors.affiliation: School of Biochemistry, University of Bristol, UK National Institute for Health Research Blood and Transplant Research Unit (NIHR BTRU) in Red Blood Cell Products, University of Bristol, UK.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Pellegrin
authors.affiliation: School of Biochemistry, University of Bristol, UK National Institute for Health Research Blood and Transplant Research Unit (NIHR BTRU) in Red Blood Cell Products, University of Bristol, UK.
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: Mordue
authors.affiliation: Department of Internal Medicine, Zuyderland Hospital, Heerlen-Sittard, The Netherlands.
authors.forename: Cees Th B M
authors.initials: CT
authors.lastname: van Deursen
authors.affiliation: Department of Internal Medicine, Zuyderland Hospital, Heerlen-Sittard, The Netherlands.
authors.forename: Nicole Heitink-Ter
authors.initials: NH
authors.lastname: Braak
authors.affiliation: Department of Hematology, University Medical Center Groningen, The Netherlands.
authors.forename: Gerwin
authors.initials: G
authors.lastname: Huls
authors.affiliation: Department of Clinical Chemistry and Hematology, Atrium Medical Center Parkstad, Heerlen, The Netherlands.
authors.forename: Mathie P G
authors.initials: MP
authors.lastname: Leers
authors.affiliation: Department of Clinical Genetics, VU University Medical Center, and Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Eline
authors.initials: E
authors.lastname: Overwater
authors.affiliation: Department of Pediatric Hematology, Beatrix Childrens Hospital, University Medical Center Groningen, The Netherlands.
authors.forename: Rienk Y J
authors.initials: RY
authors.lastname: Tamminga
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, The Netherlands.
authors.forename: Bert
authors.initials: B
authors.lastname: van der Zwaag
authors.affiliation: Oncohematology Unit - Physiopathology of Anemias Unit, Foundation IRCCS Cà Granda, Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Fermo
authors.affiliation: Oncohematology Unit - Physiopathology of Anemias Unit, Foundation IRCCS Cà Granda, Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Bianchi
authors.affiliation: Department of Clinical Chemistry and Haematology, Laboratory for Red Blood Cell Research, University Medical Center Utrecht, The Netherlands.
authors.forename: Richard
authors.initials: R
authors.lastname: van Wijk
authors.affiliation: School of Biochemistry, University of Bristol, UK National Institute for Health Research Blood and Transplant Research Unit (NIHR BTRU) in Red Blood Cell Products, University of Bristol, UK ash.m.toye@bristol.ac.uk t.satchwell@bristol.ac.uk.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Toye
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000378:metabolism; D017487:Ankyrins / Q000172:deficiency*; D001798:Blood Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism; D004900:Erythroblasts / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004920:Erythropoiesis / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D059748:Proteolysis; D013103:Spherocytosis, Hereditary / Q000235:genetics / Q000378:metabolism
chemical_list: C110202:ANK1 protein, human; D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D001798:Blood Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C498523:RHAG protein, human