title: The alpha-hemoglobin stabilizing protein and expression of unstable alpha-Hb variants.
abstract: OBJECTIVE
  To determine the role of the alpha-hemoglobin stabilizing protein (AHSP) in the clinical expression of alpha-hemoglobin (alpha-Hb) variants described as unstable, ten alpha chain variants have been studied with their chaperone. AHSP specifically binds free alpha-Hb to form a soluble heterodimer until it is replaced by the beta-Hb partner. In this way, AHSP prevents the precipitation of free alpha chains which might damage the membrane of erythrocyte. AHSP specifically recognizes the G and H helices of alpha-Hb that are also involved in the alpha1beta1 dimer interface. AHSP may act as a modifier in alpha-thalassemias and lead to the thalassemic phenotypes observed in certain unstable alpha-Hb variants previously considered unstable. The different abnormalities of the alpha chain were located either in the G helix: Hb Bronovo alpha103(G10)His-->Leu, Hb Sallanches alpha104(G11)Cys-->Tyr, Hb Oegstgeest alpha104(G11)Cys-->Ser, Hb Bleuland alpha108(G15)Thr-->Asn, Hb Suan Dok alpha109(G16)Leu-->Arg and as yet undescribed alpha109(G16)Leu-->Gln, in the GH corner: Hb Foggia alpha117(GH5)Phe-->Ser, or in the H helix: Hb Groene Hart alpha119(H2)Pro-->Ser, Hb Diamant alpha119(H2)Pro-->Leu, Hb Utrecht alpha129(H12)Leu-->Pro.
  
  
  METHODS
  These different mutated alpha-Hb were co-expressed with their chaperone AHSP as a fusion protein with glutathione S-transferase (GST) and analyzed by SDS-PAGE.
  
  
  RESULTS
  In all cases the proteins were normally synthesized in bacteria as shown by an expression level of mutated GST-alpha-Hbs similar to that observed for normal GST-alpha-Hb. In contrast, the recovered quantities of purified mutated GST-alpha-Hbs associated with AHSP are highly variable. An extreme case is GST-alpha-Hb(Utrecht) which was only found at trace levels.
  
  
  CONCLUSIONS
  One can assume that different mechanisms may be responsible for the amount of abnormal Hb recovered, such as a highly unstable alpha chain or an impaired formation of the complex AHSP/alpha-Hb or a modification of the alphabeta dimer formation.
authors.affiliation: INSERM U779, University of Paris XI, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Vasseur
authors.forename: Elisa
authors.initials: E
authors.lastname: Domingues-Hamdi
authors.forename: Thomas
authors.initials: T
authors.lastname: Brillet
authors.forename: Michael C
authors.initials: MC
authors.lastname: Marden
authors.forename: Véronique
authors.initials: V
authors.lastname: Baudin-Creuza
mesh_terms: D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility
chemical_list: C468768:AHSP protein, human; D001798:Blood Proteins; D006454:Hemoglobins; D018832:Molecular Chaperones; D050505:Mutant Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C070077:alpha(A) globin