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

Abstract

Erythrocyte precursors produce abundant alpha- and beta-globin proteins, which assemble with each other to form hemoglobin A (HbA), the major blood oxygen carrier. alphaHb-stabilizing protein (AHSP) binds free alpha 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 alpha-globin caused by beta-globin gene disruption in mice. Surprisingly, we found that AHSP also has important functions when free alpha-globin is limited. Thus, compound mutants lacking both Ahsp and 1 of 4 alpha-globin genes (genotype Ahsp(-/-)alpha-globin*(alpha/alphaalpha)) exhibited more severe anemia and Hb instability than mice with either mutation alone. In vitro, recombinant AHSP promoted folding of newly translated alpha-globin, enhanced its refolding after denaturation, and facilitated its incorporation into HbA. Moreover, in erythroid precursors, newly formed free alpha-globin was destabilized by loss of AHSP. Therefore, in addition to its previously defined role in detoxification of excess alpha-globin, AHSP also acts as a molecular chaperone to stabilize nascent alpha-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.

Figure 1. Abnormal erythrocyte morphology in Ahsp^–/–α-globin*^α/αα double-mutant mice.

Wright-Giemsa staining of peripheral blood from mice with genotypes indicated. Note that variations in erythrocyte size and shape are more severe in the compound Ahsp–α-globin mutants (far right panel). Original magnification, ×200.

Figure 2. Increased Hb instability in Ahsp^–/–α-globin*^α/αα double-mutant erythrocytes.

(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.

Figure 3. Blocked erythroid maturation in double-mutant mice.

(A) The developmental stage of splenic erythroid precursors was analyzed by flow cytometry according to a protocol described by Liu et al (31). Ter119⁺ erythroid progenitors were fractionated by forward scattering and CD71 expression into increasingly mature populations, termed Ery.A, Ery.B, and Ery.C. Results of representative experiments on wild-type and double-mutant mice are shown. (B) Two to 5 mice of each genotype were analyzed at age 6 months. The y axis shows frequencies of spleen erythroblast subsets as percentage of total Ter119⁺ cells for each genotype indicated on the x axis. Each symbol represents data for a single mouse. The horizontal bar indicates the mean value. The differences between single and double mutants were statistically significant (2-tailed P value): Ery.B: Ahsp^–/– versus Ahsp^–/–α-globin*^α/αα, P < 0.005; α-globin*^α/αα versus Ahsp^–/–α-globin*^α/αα, P < 0.001; Ery.C: Ahsp^–/– versus Ahsp^–/–α-globin*^α/αα, P < 0.08; α-globin*^α/αα versus Ahsp^–/–α-globin*^α/αα, P < 0.003.

Figure 4. AHSP stabilizes newly synthesized α-globin in vitro.

(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.

Figure 5. AHSP facilitates α-globin folding.

(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.

Figure 6. Identification of transient free αHb and αHb-AHSP complex in erythroid cells.

Reticulocytes from β thalassemia intermedia mice (strain th-3) with wild-type or mutant Ahsp alleles were pulse-labeled with ³⁵S-methionine for 10 minutes, lysed, and then treated with carbon monoxide to stabilize newly synthesized Hbs. Soluble proteins were fractionated by isoelectric focusing and visualized by autoradiography (top panel). Purified human βHb (hβHb; 100 μg/ml) or recombinant mouse AHSP (500 μg/ml) were added separately to different aliquots of each sample 30 minutes prior to electrophoresis, as indicated. The identities of the major bands are shown at the left. Free αHb (prominently visible in lanes 1 and 4) was observed at relatively high level in β thalassemic mice and to a lesser extent in wild-type mice (data not shown); this band shifted to the position of a human-mouse Hb heterotetramer (mα₂hβ₂) upon addition of human βHb (lanes 2 and 5) and to the position of a putative αHb-AHSP complex upon addition of purified recombinant AHSP (lanes 3 and 6). Note that in Ahsp^–/– reticulocytes, the free αHb band was markedly reduced and the αHb-AHSP band was absent (lanes 7 and 8). The identity of the slowest migrating band (asterisk) is unknown. The bottom panel shows the same unstained CAE membrane with steady-state HbA chains visible. Note that free αHb and αHb-AHSP complex were not visualized, indicating that these species are relatively short-lived.

Figure 7. AHSP stabilizes multiple forms of α-globin at different stages of HbA synthesis and homeostasis.

Prior studies showed that AHSP (shown in red) inhibits ROS production and precipitation from excess αHb that accumulates in β thalassemia. The current study demonstrates that AHSP (shown in blue) acts as a molecular chaperone to promote native folding and stability of apo-α-globin and αHb en route to HbA synthesis. The brown circle indicates oxidized αHb.