Hideaki Moriyama

Evolutionary and functional insights into the mechanism underlying high-altitude adaptation of deer mouse hemoglobin

Adaptive modifications of heteromeric proteins may involve genetically based changes in single subunit polypeptides or parallel changes in multiple genes that encode distinct, interacting subunits. Here we investigate these possibilities by conducting a combined evolutionary and functional analysis of duplicated globin genes in natural populations of deer mice (Peromyscus maniculatus) that are adapted to different elevational zones. A multilocus analysis of nucleotide polymorphism and linkage disequilibrium revealed that high-altitude adaptation of deer mouse hemoglobin involves parallel functional differentiation at multiple unlinked gene duplicates: two α-globin paralogs on chromosome 8 and two β-globin paralogs on chromosome 1. Differences in O2-binding affinity of the alternative β-chain hemoglobin isoforms were entirely attributable to allelic differences in sensitivity to 2,3-diphosphoglycerate (DPG), an allosteric cofactor that stabilizes the low-affinity, deoxygenated conformation of the hemoglobin tetramer. The two-locus β-globin haplotype that predominates at high altitude is associated with suppressed DPG-sensitivity (and hence, increased hemoglobin-O2 affinity), which enhances pulmonary O2 loading under hypoxia. The discovery that allelic differences in DPG-sensitivity contribute to adaptive variation in hemoglobin–O2 affinity illustrates the value of integrating evolutionary analyses of sequence variation with mechanistic appraisals of protein function. Investigation into the functional significance of the deer mouse β-globin polymorphism was motivated by the results of population genetic analyses which revealed evidence for a history of divergent selection between elevational zones. The experimental measures of O2-binding properties corroborated the tests of selection by demonstrating a functional difference between the products of alternative alleles.

Epistasis among adaptive mutations in deer mouse hemoglobin.

Holding Your Breath Hemoglobin and myoglobin are widely responsible for oxygen transport and storage (see the Perspective by Rezende). The ability of diving mammals to obtain enough oxygen to support extended dives and foraging is largely dependent on muscle myoglobin (Mb) content. Mirceta et al. (p. 1303) found that in mammalian lineages with an aquatic or semiaquatic lifestyle, Mb net charge increases, which may represent an adaptation to inhibit self-association of Mb at high intracellular concentrations. Epistasis results from nonadditive genetic interactions and can affect phenotypic evolution. Natarajan et al. (p. 1324) found that epistatic interactions were able to explain the increased hemoglobin oxygen-binding affinity observed in deer mice populations at high altitude. In mammals, the offloading of oxygen from hemoglobin is facilitated by a reduction in the bloods pH, driven by metabolically produced CO2. However, in fish, a reduction in blood pH reduces oxygen carrying capacity of hemoglobin. Rummer et al. (p. 1327) implanted fiber optic oxygen sensors within the muscles of rainbow trout and found that elevated CO2 levels in the water led to acidosis and elevated oxygen tensions. Deer mice have discovered that mutations distant from the oxygen-binding site help them live at high altitude. [Also see Perspective by Rezende] Epistatic interactions between mutant sites in the same protein can exert a strong influence on pathways of molecular evolution. We performed protein engineering experiments that revealed pervasive epistasis among segregating amino acid variants that contribute to adaptive functional variation in deer mouse hemoglobin (Hb). Amino acid mutations increased or decreased Hb-O2 affinity depending on the allelic state of other sites. Structural analysis revealed that epistasis for Hb-O2 affinity and allosteric regulatory control is attributable to indirect interactions between structurally remote sites. The prevalence of sign epistasis for fitness-related biochemical phenotypes has important implications for the evolutionary dynamics of protein polymorphism in natural populations.

Mechanisms of Hemoglobin Adaptation to High Altitude Hypoxia

Evidence from a number of vertebrate taxa suggests that modifications of hemoglobin (Hb) function may often play a key role in mediating an adaptive response to high altitude hypoxia. The respiratory functions of Hb are a product of the proteins intrinsic O(2)-binding affinity and its interactions with allosteric effectors such as protons, chloride ions, CO(2), and organic phosphates. Here we review several case studies involving high altitude vertebrates where it has been possible to identify specific mechanisms of Hb adaptation to hypoxia. In addition to comparative studies of Hbs from diverse animal species, functional studies of human Hb mutants also suggest that there is ample scope for evolutionary adjustments in Hb-O(2) affinity through alterations of the equilibrium constants of O(2) binding to deoxy- and oxyHb or through changes in the allosteric equilibrium constants for the transition between the deoxy- and oxyHb quaternary structures. It may be the case that certain evolutionary paths are followed more often than others simply because they are subject to less stringent pleiotropic constraints.

Genetic differences in hemoglobin function between highland and lowland deer mice

SUMMARY In high-altitude vertebrates, adaptive changes in blood–O2 affinity may be mediated by modifications of hemoglobin (Hb) structure that affect intrinsic O2 affinity and/or responsiveness to allosteric effectors that modulate Hb–O2 affinity. This mode of genotypic specialization is considered typical of mammalian species that are high-altitude natives. Here we investigated genetically based differences in Hb–O2 affinity between highland and lowland populations of the deer mouse (Peromyscus maniculatus), a generalist species that has the broadest altitudinal distribution of any North American mammal. The results of a combined genetic and proteomic analysis revealed that deer mice harbor a high level of Hb isoform diversity that is attributable to allelic polymorphism at two tandemly duplicated α-globin genes and two tandemly duplicated β-globin genes. This high level of isoHb diversity translates into a correspondingly high level of interindividual variation in Hb functional properties. O2 equilibrium experiments revealed that the Hbs of highland mice exhibit slightly higher intrinsic O2 affinities and significantly lower Cl– sensitivities relative to the Hbs of lowland mice. The experiments also revealed distinct biochemical properties of deer mouse Hb related to the anion-dependent allosteric regulation of O2 affinity. In conjunction with previous findings, our results demonstrate that modifications of Hb structure that alter allosteric anion sensitivity play an important role in the adaptive fine-tuning of blood–O2 affinity.

Repeated elevational transitions in hemoglobin function during the evolution of Andean hummingbirds

Significance Hummingbirds have exceedingly high oxygen demands because of their elevated rates of aerobic metabolism, and yet they thrive in high-altitude environments in the Andes where oxygen is scarce. Here we report the finding that when hummingbird species colonized new elevational zones, evolutionary changes in the respiratory properties of hemoglobin were repeatedly mediated by the same amino acid replacements. Specifically, ancestral sequence reconstruction and protein engineering experiments revealed that parallel adaptation of hemoglobin function in multiple species is attributable to repeated amino acid replacements at a single pair of interacting sites. This striking parallelism at the molecular level suggests a surprising degree of reproducibility and predictability in adaptive protein evolution. Animals that sustain high levels of aerobic activity under hypoxic conditions (e.g., birds that fly at high altitude) face the physiological challenge of jointly optimizing blood-O2 affinity for O2 loading in the pulmonary circulation and O2 unloading in the systemic circulation. At high altitude, this challenge is especially acute for small endotherms like hummingbirds that have exceedingly high mass-specific metabolic rates. Here we report an experimental analysis of hemoglobin (Hb) function in South American hummingbirds that revealed a positive correlation between Hb-O2 affinity and native elevation. Protein engineering experiments and ancestral-state reconstructions revealed that this correlation is attributable to derived increases in Hb-O2 affinity in highland lineages, as well as derived reductions in Hb-O2 affinity in lowland lineages. Site-directed mutagenesis experiments demonstrated that repeated evolutionary transitions in biochemical phenotype are mainly attributable to repeated amino acid replacements at two epistatically interacting sites that alter the allosteric regulation of Hb-O2 affinity. These results demonstrate that repeated changes in biochemical phenotype involve parallelism at the molecular level, and that mutations with indirect, second-order effects on Hb allostery play key roles in biochemical adaptation.

Mitochondrial genome dynamics in plants and animals: convergent gene fusions of a MutS homologue.

Mitochondrial processes influence a broad spectrum of physiological and developmental events in higher eukaryotes, and their aberrant function can lead to several familiar disease phenotypes in mammals. In plants, mitochondrial genes directly influence pollen development and the occurrence of male sterility in natural plant populations. Likewise, in animal systems evidence accumulates to suggest important mitochondrial functions in spermatogenesis and reproduction. Here we present evidence for a convergent gene fusion involving a MutS-homologous gene functioning within the mitochondrion and designated Msh1. In only plants and soft corals, the MutS homologue has fused with a homing endonuclease sequence at the carboxy terminus of the protein. However, the endonuclease domains in the plants and the soft corals are members of different groups. In plants, Msh1 can influence mitochondrial genome organization and male sterility expression. Based on parallels in Msh1 gene structure shared by plants and corals, and their similarities in reproductive behavior, we postulate that this convergent gene fusion might have occurred in response to coincident adaptive pressures on reproduction.

Epistasis Constrains Mutational Pathways of Hemoglobin Adaptation in High-Altitude Pikas

A fundamental question in evolutionary genetics concerns the roles of mutational pleiotropy and epistasis in shaping trajectories of protein evolution. This question can be addressed most directly by using site-directed mutagenesis to explore the mutational landscape of protein function in experimentally defined regions of sequence space. Here, we evaluate how pleiotropic trade-offs and epistatic interactions influence the accessibility of alternative mutational pathways during the adaptive evolution of hemoglobin (Hb) function in high-altitude pikas (Mammalia: Lagomorpha). By combining ancestral protein resurrection with a combinatorial protein-engineering approach, we examined the functional effects of sequential mutational steps in all possible pathways that produced an increased Hb–O2 affinity. These experiments revealed that the effects of mutations on Hb–O2 affinity are highly dependent on the temporal order in which they occur: Each of three β-chain substitutions produced a significant increase in Hb–O2 affinity on the ancestral genetic background, but two of these substitutions produced opposite effects when they occurred as later steps in the pathway. The experiments revealed pervasive epistasis for Hb–O2 affinity, but affinity-altering mutations produced no significant pleiotropic trade-offs. These results provide insights into the properties of adaptive substitutions in naturally evolved proteins and suggest that the accessibility of alternative mutational pathways may be more strongly constrained by sign epistasis for positively selected biochemical phenotypes than by antagonistic pleiotropy.

Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 Å resolution

The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild‐type RNase A and three mutants—F120A, F120G, and F120W—were analyzed up to a 1.4 Å resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild‐type RNase A, except for the C‐terminal region of F120G with a high B‐factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild‐type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild‐type RNase A, and those of F120W and F120G appeared to be “inside out” compared with that of wild‐type RNase A. Only ∼1 Å change in the distance between Nε2 of His12 and Nδ1 of His119 causes a drastic decrease in kcat, indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.

Convergent Evolution of Hemoglobin Function in High-Altitude Andean Waterfowl Involves Limited Parallelism at the Molecular Sequence Level

A fundamental question in evolutionary genetics concerns the extent to which adaptive phenotypic convergence is attributable to convergent or parallel changes at the molecular sequence level. Here we report a comparative analysis of hemoglobin (Hb) function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergence in the oxygenation properties of Hb, and to assess the extent to which convergence in biochemical phenotype is attributable to repeated amino acid replacements. Functional experiments on native Hb variants and protein engineering experiments based on site-directed mutagenesis revealed the phenotypic effects of specific amino acid replacements that were responsible for convergent increases in Hb-O2 affinity in multiple high-altitude taxa. In six of the eight taxon pairs, high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique replacements, parallel replacements (involving identical-by-state variants with independent mutational origins in different lineages), and collateral replacements (involving shared, identical-by-descent variants derived via introgressive hybridization). In genome scans of nucleotide differentiation involving high- and low-altitude populations of three separate species, function-altering amino acid polymorphisms in the globin genes emerged as highly significant outliers, providing independent evidence for adaptive divergence in Hb function. The experimental results demonstrate that convergent changes in protein function can occur through multiple historical paths, and can involve multiple possible mutations. Most cases of convergence in Hb function did not involve parallel substitutions and most parallel substitutions did not affect Hb-O2 affinity, indicating that the repeatability of phenotypic evolution does not require parallelism at the molecular level.

Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds

Background: The functional significance of hemoglobin heterogeneity remains a mystery. Results: In adult birds, the HbD isoform (related to embryonic hemoglobin) exhibits distinct oxygenation properties relative to the major HbA isoform. Conclusion: Substitutions that distinguish HbD from HbA are not shared with embryonic hemoglobin. Significance: Differences between isoforms stem from derived (nonancestral) changes in duplicated genes, not from the retention of an ancestral condition. 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 αA-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the αD-globin gene). The αD-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 O2 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-O2 affinity stems primarily from changes in the O2 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 αD-globin gene that is shared with the embryonic α-like globin gene.