title: Investigating the key membrane protein changes during in vitro erythropoiesis of protein 4.2 (-) cells (mutations Chartres 1 and 2).
abstract: BACKGROUND
  Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47, CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex.
  
  
  METHODS
  We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis.
  
  
  RESULTS
  We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47, higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression, arguing against a direct reciprocal relationship.
  
  
  CONCLUSIONS
  We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated, in part, by increased CD44-cytoskeleton binding.
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University Walk, Bristol, UK.
authors.forename: Emile
authors.initials: E
authors.lastname: van den Akker
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Satchwell
authors.forename: Stephanie
authors.initials: S
authors.lastname: Pellegrin
authors.forename: Joanna F
authors.initials: JF
authors.lastname: Flatt
authors.forename: Michel
authors.initials: M
authors.lastname: Maigre
authors.forename: Geoff
authors.initials: G
authors.lastname: Daniels
authors.forename: Jean
authors.initials: J
authors.lastname: Delaunay
authors.forename: Lesley J
authors.initials: LJ
authors.lastname: Bruce
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Toye
mesh_terms: D000328:Adult; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D017487:Ankyrins / Q000378:metabolism; D001483:Base Sequence; D001798:Blood Proteins / Q000235:genetics / Q000378:metabolism; D051928:CD47 Antigen / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004920:Erythropoiesis; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D001798:Blood Proteins; D051928:CD47 Antigen; D003598:Cytoskeletal Proteins; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C498523:RHAG protein, human; C044791:erythrocyte membrane band 4.2 protein