Gene duplication and the evolution of hemoglobin isoform differentiation in birds

Abstract

The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the α(A)-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the α(D)-globin gene). The α(D)-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O(2) affinity in the presence of allosteric effectors such as organic phosphates and Cl(-) ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O(2) affinity stems primarily from changes in the O(2) association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the α(D)-globin gene that is shared with the embryonic α-like globin gene.

FIGURE 1.

Hypothetical scenarios depicting the phylogenetic distribution of amino acid substitutions that are responsible for functional differentiation between the co-expressed HbA and HbD isoforms in birds. The phylogeny represented in each panel depicts the known branching relationships among the α^A-, α^D-, and α^E-globin genes. At any given site, fixed differences between the α^A- and α^D-globin genes could be attributable to a substitution that occurred on the branch leading to α^A-globin (A), a substitution that occurred on the post-duplication branch leading to α^D-globin (B), substitutions that occurred on the branch leading to α^A-globin and on the post-duplication branch leading to α^D-globin (C), a substitution that occurred on the pre-duplication branch leading to the single-copy progenitor of α^D- and α^E-globin (D), substitutions that occurred on the branch leading to α^A-globin and on the pre-duplication branch leading to the α^D/α^E ancestor (E), or substitutions that occurred on the pre-duplication branch leading to the α^D/α^E ancestor and on the post-duplication branch leading to α^D-globin (F).

FIGURE 2.

O₂ affinity and cooperativity (P₅₀ and n₅₀, respectively) of pheasant HbA and HbD as a function of pH, temperature, and in the absence and presence of IHP (IHP/H4_B ratio = 23.5). O₂ equilibria were measured in 0.1 m NaHEPES buffer containing 0.1 m KCl. Heme concentration, 0.08 mm (HbA) and 0.11 mm (HbD) and 0.10 (HbA +D).

FIGURE 3.

Extended Hill plots of O₂ equilibria (where Y = fractional O₂ saturation) for pheasant HbA and HbD. A, HbA and HbD at 25 °C; B, HbD at 25 and 37 °C and in the absence and presence of saturating IHP concentration (IHP/Hb ratio = 23.5). In each plot, the intercept of the lower asymptote with the horizontal line at logY/(Y − 1) = 0 provides an estimate of K_T, the O₂ association constant of T-state deoxy-Hb, and the intercept of the upper asymptote with the same line provides an estimate of K_R, the O₂ association constant of R-state oxyHb. Heme concentration 0.60 (HbA and HbD); other conditions are as described in the legend for Fig. 2.

FIGURE 4.

Adair constants (k₁, k₂, k₃, and k₄) for pheasant HbA and HbD as a function of temperature, pH, and the absence and presence of IHP (derived from data shown in Fig. 2).

FIGURE 5.

Reconstructed ancestral states of 39 sites that distinguish the α^A- and α^D-globin polypeptides. As shown in the inset phylogeny of α-like globin genes, ancestral states for each of the 39 sites were reconstructed for four separate nodes in the tree.

FIGURE 6.

Homology-based structural models of pheasant HbA and HbD showing predicted differences in the stereochemistry of IHP binding between the N and C termini of the α-chain subunits.