Biology subjects

Fago, A.

Publications and source records attributed to Fago, A..

6 recordsLinked to original sources

bioRxiv2022

Evolution of an extreme hemoglobin phenotype contributed to the sub-Arctic specialization of extinct Steller's sea cows

The extinct Stellers sea cow (Hydrodamalis gigas; {dagger}1768) was a whale-sized marine mammal that manifested profound morphological specializations to exploit the harsh coastal climate of the North Pacific. Yet despite first-hand accounts of their biology, little is known regarding the physiological adjustments underlying their evolution to this environment. Here, the adult-expressed hemoglobin (Hb; 2{beta}/{delta}2) of this sirenian is shown to harbor a fixed amino acid replacement at an otherwise invariant position ({beta}/{delta}82Lys[->]Asn) that alters multiple aspects of Hb function. First, our functional characterization of recombinant sirenian Hb proteins demonstrate that the Hb-O2 affinity of this sub-Arctic species was less affected by temperature than those of living (sub)tropical sea cows. This phenotype presumably safeguarded O2 delivery to cool peripheral tissues and largely arises from a reduced intrinsic temperature sensitivity of the H. gigas protein. Additional experiments on H. gigas {beta}/{delta}82Asn[->]Lys mutant Hb further reveal this exchange renders Stellers sea cow Hb unresponsive to the potent intraerythrocytic allosteric effector 2,3-diphosphoglycerate, a radical modification that is the first documented example of this phenotype among mammals. Notably, {beta}/{delta}82Lys[->]Asn moreover underlies the secondary evolution of a reduced blood-O2 affinity phenotype that would have promoted heightened tissue and maternal/fetal O2 delivery. This conclusion is bolstered by analyses of two Stellers sea cow prenatal Hb proteins (Hb Gower I; {zeta}2{varepsilon}2 and HbF; 2{gamma}2) that suggest an exclusive embryonic stage expression pattern, and reveal uncommon replacements in H. gigas HbF ({gamma}38Thr[->]Ile and {gamma}101Glu[->]Asp) that increased Hb-O2 affinity relative to dugong HbF. Finally, the {beta}/{delta}82Lys[->]Asn replacement of the adult/fetal protein is shown to increase protein solubility, which may have elevated red blood cell Hb content within both the adult and fetal circulations and contributed to meeting the elevated metabolic (thermoregulatory) requirements and fetal growth rates associated with this species cold adaptation.

evolutionary biology↗

bioRxiv2022

Evolution and molecular basis of a novel allosteric property of crocodilian hemoglobin

Understanding the evolution of novel protein functions requires data on the mechanistic effects of causative mutations and the extent of coupling between the gain of new function and loss of ancestral function. Here, we use ancestral protein resurrection and directed mutagenesis to dissect the molecular basis of a novel mode of allosteric regulation in crocodilian hemoglobin. We discovered that regulation of Hb-O2 affinity via allosteric binding of bicarbonate ions (a biochemical adaptation unique to crocodilians) and the concomitant loss of allosteric regulation by ATP-binding are attributable to non-overlapping sets of substitutions. Gain of bicarbonate-sensitivity involved direct effects of few replacements at key sites in combination with indirect effects of numerous replacements at structurally disparate sites. Due to the context-dependence of causative substitutions, the unique allosteric properties of crocodilian hemoglobin cannot be easily transplanted into divergent homologs of other species. Significance StatementThe extraordinary breath-hold diving capacity of crocodilians is partly attributable to a unique mode of allosterically regulating hemoglobin-oxygenation in circulating red blood cells. We investigated the origin and mechanistic basis of this novel biochemical adaptation by performing directed mutagenesis experiments on resurrected ancestral crocodilian hemoglobins. Our results revealed that evolved changes in allosteric regulation involved the direct effect of few amino acid substitutions at key sites in combination with indirect effects of numerous other substitutions at structurally disparate sites. Such indirect interaction effects suggest that the evolution of novel protein functions may often depend on neutral mutations that produce no adaptive benefit when they first arise, but which contribute to a permissive background for subsequent function-altering mutations at other sites.

evolutionary biology↗

bioRxiv2021

New insights into the allosteric effects of CO2 and bicarbonate on crocodilian hemoglobin

Crocodilians are unique among vertebrates in that their hemoglobin (Hb) O2 binding is allosterically regulated by bicarbonate, which forms in the red blood cell upon hydration of CO2. Although known for decades, this remarkable mode of allosteric control has not yet been experimentally verified with direct evidence of bicarbonate binding to crocodilian Hb, probably because of confounding CO2-mediated effects. Here we provide the first quantitative analysis of the separate allosteric effects of CO2 and bicarbonate on Hb of the spectacled caiman (Caiman crocodilus). Using thin-layer gas diffusion chamber and Tucker chamber techniques, we demonstrate that both CO2 and bicarbonate bind to Hb with high affinity and strongly decrease Hb-O2 saturation, and propose that both effectors bind to an unidentified positively charged site containing a reactive amino group in the low-O2 affinity T conformation of the Hb. These results provide the first experimental evidence that bicarbonate binds directly to crocodilian Hb and promotes O2 delivery independently of CO2. Using the gas-diffusion chamber, we observed similar effects in the Hbs of a phylogenetically diverse set of other caiman, alligator, and crocodile species, suggesting that the unique mode of allosteric regulation by CO2 and bicarbonate evolved >80-100 million years ago in the common ancestor of crocodilians. Taken together, our results show a tight and unusual linkage between O2 and CO2 transport in the blood of crocodilians, where build-up of blood CO2 and bicarbonate ions during breath-hold diving or digestion facilitates O2 delivery, while Hb desaturation facilitates CO2 transport as protein-bound CO2 and bicarbonate.

physiology↗

bioRxiv2021

Genetic variation in haemoglobin regulates breathing in high-altitude deer mice

Physiological systems often have emergent properties but the effects of genetic variation on physiology are often unknown, which presents a major challenge to understanding the mechanisms of phenotypic evolution. We investigated the in vivo effects on respiratory physiology of genetic variants in haemoglobin (Hb) that contribute to hypoxia adaptation in high-altitude deer mice (Peromyscus maniculatus). We created F2 inter-population hybrids of highland and lowland deer mice to test the phenotypic effects of - and {beta}-globin variants on a mixed genetic background. High-altitude genotypes were associated with breathing phenotypes that enhance O2 uptake in hypoxia, including a deeper more effective breathing pattern and an augmented hypoxic ventilatory response. These effects could not be explained by erythrocyte Hb-O2 affinity or globin gene expression in the brainstem. Therefore, adaptive variation in haemoglobin can have unexpected effects on physiology that are distinct from the canonical function of this protein in circulatory O2 transport.

evolutionary biology↗

bioRxiv2019

Oxygenation properties of hemoglobin and the evolutionary origins of isoform multiplicity in an amphibious air-breathing fish, the blue-spotted mudskipper (Boleophthalmus pectinirostris)

Among the numerous lineages of teleost fish that have independently transitioned from obligate water-breathing to facultative air-breathing, evolved properties of hemoglobin (Hb)-O2 transport may have been shaped by the prevalence and severity of aquatic hypoxia (which influences the extent to which fish are compelled to switch to aerial respiration) as well as the anatomical design of air-breathing structures and the cardiovascular system. Here we examine the structure and function of Hbs in an amphibious, facultative air-breathing fish, the blue-spotted mudskipper (Boleophthalmus pectinirostris). We also characterized the genomic organization of the globin gene clusters of the species and we integrated phylogenetic and comparative genomic analyses to unravel the duplicative history of the genes that encode the subunits of structurally distinct mudskipper Hb isoforms (isoHbs). The B. pectinirostris isoHbs exhibit high intrinsic O2-affinities, similar to those of hypoxia-tolerant, water-breathing teleosts, and remarkably large Bohr effects. Genomic analysis of conserved synteny revealed that the genes that encode the -type subunits of the two main adult isoHbs are members of paralogous gene clusters that represent products of the teleost-specific whole-genome duplication. Experiments revealed no appreciable difference in the oxygenation properties of co-expressed isoHbs in spite of extensive amino acid divergence between the alternative -chain subunit isoforms. It therefore appears that the ability to switch between aquatic and aerial respiration does not necessarily require a division of labor between functionally distinct isoHbs with specialized oxygenation properties. Summary statementThe blue-spotted mudskipper routinely switches between aquatic and aerial respiration. This respiratory versatility is associated with properties of hemoglobin-oxygen transport that are similar to those found in hypoxia-adapted water-breathing fishes.

physiology↗

bioRxiv2017

Molecular basis of hemoglobin adaptation in the high-flying bar-headed goose

During adaptive phenotypic evolution, some selectively fixed mutations may be directly causative and others may be purely compensatory. The relative contribution of these two classes of mutation depends on the form and prevalence of mutational pleiotropy. To investigate the nature of adaptive substitutions and their pleiotropic effects, we used a protein engineering approach to characterize the molecular basis of hemoglobin (Hb) adaptation in the bar-headed goose (Anser indicus), a hypoxia-tolerant species renowned for its trans-Himalayan migratory flights. We synthesized and tested all possible mutational intermediates in the line of descent connecting the wildtype bar-headed goose genotype with the most recent common ancestor of bar-headed goose and its lowland relatives. Site-directed mutagenesis experiments revealed effect-size distributions of causative mutations and biophysical mechanisms underlying changes in function. Trade-offs between alternative functional properties revealed the importance of compensating deleterious pleiotropic effects in the adaptive evolution of protein function.

genomics↗