OBJECTIVES: 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. DESIGN AND 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. CONCLUSION: 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.
OBJECTIVES: 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. DESIGN AND 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. CONCLUSION: 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: Christopher S Thom; Claire F Dickson; David A Gell; Mitchell J Weiss Journal: Cold Spring Harb Perspect Med Date: 2013-03-01 Impact factor: 6.915