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 2

Increased Hb instability in Ahsp–/–α-globin*α/αα double-mutant erythrocytes.

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Increased Hb instability in Ahsp–/–α-globin*α/αα double-mutant erythrocy...
(A) Steady-state membrane-associated Hbs. TAU gel analysis of membrane skeletons prepared from equal numbers of erythrocytes from mice with genotypes indicated. α- and β-globin chains were stained with Coomassie blue and are indicated. Lane 9 represents an altered β-globin genotype, which results in 2 bands. (B) Analysis of newly synthesized globin chains. Equal numbers of reticulocytes were pulse-labeled with 35S-methionine and 35S-cysteine for 15 minutes, then disrupted by hypotonic lysis. Ahsp and α-globin genotypes are indicated. Soluble cytoplasmic (C) and membrane-associated (M) globins were isolated by differential centrifugation, fractionated on TAU gels, and visualized by autoradiography. The data shown derive from mice with the diffuse β-globin genotype. Results of quantitative analysis of nascent globins in membrane and cytoplasmic fractions from multiple experiments are shown in Table 2. (C) Quantification of ROS. Erythrocytes were incubated with dichlorofluorescin diacetate (DCFH-DA), which enters cells and is converted by ROS to the fluorescent product DCF. Representative flow cytometry data from 2 mice of each genotype are shown. Data from all mice analyzed are summarized in the upper-right corner of each panel, which shows the mean fluorescent intensity of DCF signal with wild-type erythrocytes normalized to 1.0. Three to 6 mice from each group were analyzed.