An erythroid chaperone that facilitates folding of α-globin subunits for hemoglobin synthesis
Xiang Yu, … , Andrew J. Gow, Mitchell J. Weiss
Xiang Yu, … , Andrew J. Gow, Mitchell J. Weiss
Published July 2, 2007
Citation Information: J Clin Invest. 2007;117(7):1856-1865. https://doi.org/10.1172/JCI31664.
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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 5

AHSP facilitates α-globin folding.

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AHSP facilitates α-globin folding. (A) AHSP confers protease resistance ...
(A) AHSP confers protease resistance to nascent apo-α-globin. 35S-radiolabeled α-globin was synthesized by TNT using wheat germ extract with or without 4 μg/ml recombinant AHSP or CN-hemin (0.2 μM), then treated with 10 μg/ml trypsin for the indicated times. Polypeptides were fractionated on SDS-PAGE gels and visualized by autoradiography. (B) Quantitative analysis of signal intensities for full-length α-globin from the experiment represented in A. (C) Reduced α-globin–binding mutant AHSP D43R fails to confer protease resistance to nascent a-globin. The experiment was performed as described for Figure 5A. (D) Quantitative analysis of signal intensities for full-length α-globin from the experiment represented in C. (E) AHSP promotes refolding of denatured apo-α-globin. Denatured apo-α-globin (5 μM) and purified recombinant AHSP (9 μM) were analyzed for α-helical content by CD separately and then mixed together, incubated for 30 minutes at 4°C, and reanalyzed. Helical content is indicated by a negative peak in the ellipticity signal at 222 nm. Note that the measured helical content of the AHSP plus apo-α-globin mixture (labeled “observed”) exceeds the calculated theoretical sum of the helical content of AHSP and apo-α-globin measured separately. (F) Difference spectra (observed — theoretical CD spectra) for various α-globin–AHSP mixtures. The spectra shown are derived from B and from Supplemental Figure 2, E and F.