The α-hemoglobin stabilizing protein and expression of unstable α-Hb variants

ObjectivesTo determine the role of the alpha-hemoglobin stabilizing protein (AHSP) in the clinical expression of α-hemoglobin (α-Hb) variants described as unstable, ten α chain variants have been studied with their chaperone. AHSP specifically binds free α-Hb to form a soluble heterodimer until it is replaced by the ß-Hb partner. In this way, AHSP prevents the precipitation of free α chains which might damage the membrane of erythrocyte. AHSP specifically recognizes the G and H helices of α-Hb that are also involved in the α1ß1 dimer interface. AHSP may act as a modifier in α-thalassemias and lead to the thalassemic phenotypes observed in certain unstable α-Hb variants previously considered unstable.The different abnormalities of the α chain were located either in the G helix: Hb Bronovo α103(G10)His → Leu, Hb Sallanches α104(G11)Cys → Tyr, Hb Oegstgeest α104(G11)Cys → Ser, Hb Bleuland α108(G15)Thr → Asn, Hb Suan Dok α109(G16)Leu → Arg and as yet undescribed α109(G16)Leu → Gln, in the GH corner: Hb Foggia α117(GH5)Phe → Ser, or in the H helix: Hb Groene Hart α119(H2)Pro → Ser, Hb Diamant α119(H2)Pro → Leu, Hb Utrecht α129(H12)Leu → Pro.Design and methodsThese different mutated α-Hb were co-expressed with their chaperone AHSP as a fusion protein with glutathione S-transferase (GST) and analyzed by SDS-PAGE.ResultsIn all cases the proteins were normally synthesized in bacteria as shown by an expression level of mutated GST-α-Hbs similar to that observed for normal GST-α-Hb. In contrast, the recovered quantities of purified mutated GST-α-Hbs associated with AHSP are highly variable. An extreme case is GST-α-HbUtrecht which was only found at trace levels.ConclusionOne 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/α-Hb or a modification of the αβ dimer formation.