An erythroid chaperone that facilitates folding of α-globin subunits for hemoglobin synthesis
Xiang Yu, Yi Kong, Louis C. Dore, Osheiza Abdulmalik, Anne M. Katein, Suiping Zhou, John K. Choi, David Gell, Joel P. Mackay, Andrew J. Gow, Mitchell J. Weiss
Xiang Yu, Yi Kong, Louis C. Dore, Osheiza Abdulmalik, Anne M. Katein, Suiping Zhou, John K. Choi, David Gell, Joel P. Mackay, Andrew J. Gow, Mitchell J. Weiss
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Research Article

An erythroid chaperone that facilitates folding of α-globin subunits for hemoglobin synthesis

Abstract

Erythrocyte precursors produce abundant α- and β-globin proteins, which assemble with each other to form hemoglobin A (HbA), the major blood oxygen carrier. αHb-stabilizing protein (AHSP) binds free α subunits reversibly to maintain their structure and limit their ability to generate reactive oxygen species. Accordingly, loss of AHSP aggravates the toxicity of excessive free α-globin caused by β-globin gene disruption in mice. Surprisingly, we found that AHSP also has important functions when free α-globin is limited. Thus, compound mutants lacking both Ahsp and 1 of 4 α-globin genes (genotype Ahsp–/–α-globin*α/αα) exhibited more severe anemia and Hb instability than mice with either mutation alone. In vitro, recombinant AHSP promoted folding of newly translated α-globin, enhanced its refolding after denaturation, and facilitated its incorporation into HbA. Moreover, in erythroid precursors, newly formed free α-globin was destabilized by loss of AHSP. Therefore, in addition to its previously defined role in detoxification of excess α-globin, AHSP also acts as a molecular chaperone to stabilize nascent α-globin for HbA assembly. Our findings illustrate what we believe to be a novel adaptive mechanism by which a specialized cell coordinates high-level production of a multisubunit protein and protects against various synthetic imbalances.

Authors

Xiang Yu, Yi Kong, Louis C. Dore, Osheiza Abdulmalik, Anne M. Katein, Suiping Zhou, John K. Choi, David Gell, Joel P. Mackay, Andrew J. Gow, Mitchell J. Weiss

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Figure 4

AHSP stabilizes newly synthesized α-globin in vitro.

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AHSP stabilizes newly synthesized α-globin in vitro. (A) Experimental ap...
(A) Experimental approach. α-Globin was synthesized in a wheat germ–based TNT system containing 35S-methionine and heme, with or without purified recombinant AHSP protein added. Then, cold βHb was added, and HbA tetramer (α2β2) formation was assessed by CAE and autoradiography. α cDNA, α-globin cDNA. (B) Dose-response effects of added AHSP on subsequent HbA formation from newly synthesized α-globin. (C) Effect of varying the time of addition of recombinant AHSP (50 ng) to the α-globin TNT reaction. AHSP was added at 0, 20, 40, or 60 minutes after initiation of 35S-labeled α-globin synthesis. At 60 minutes, purified βHb was added and formation of HbA examined by CAE. α-Globin was most effectively utilized for HbA formation when AHSP was present at the beginning of the TNT reaction. (D) 35S-labeled α-globin was synthesized with added AHSP D43R, a reduced-affinity α-globin–binding mutant. In contrast to wild-type AHSP, the D43R mutant does not augment formation of HbA from of TNT-derived α-globin.