An official website of the United States government
Hemoglobins (Hbs) of crocodilians are reportedly characterized by unique mechanisms of allosteric regulatory control, but there are conflicting reports regarding the importance of different effectors, such as chloride ions, organic phosphates, and CO 2 . Progress in understanding the unusual properties of crocodilian Hbs has also been hindered by a dearth of structural information. Here, we present the first comparative analysis of blood properties and Hb structure and function in a phylogenetically diverse set of crocodilian species. We examine mechanisms of allosteric regulation in the Hbs of 13 crocodilian species belonging to the families Crocodylidae and Alligatoridae. We also report new amino acid sequences for the α- and β-globins of these taxa, which, in combination with structural analyses, provide insights into molecular mechanisms of allosteric regulation. All crocodilian Hbs exhibited a remarkably strong sensitivity to CO 2 , which would permit effective O 2 unloading to tissues in response to an increase in metabolism during intense activity and diving. Although the Hbs of all crocodilians exhibit similar intrinsic O 2 -affinities, there is considerable variation in sensitivity to Cl − ions and ATP, which appears to be at least partly attributable to variation in the extent of NH 2 -terminal acetylation. Whereas chloride appears to be a potent allosteric effector of all crocodile Hbs, ATP has a strong, chloride-independent effect on Hb-O 2 affinity only in caimans. Modeling suggests that allosteric ATP binding has a somewhat different structural basis in crocodilian and mammalian Hbs.
more »
« less
Effect of NH2-terminal acetylation on the oxygenation properties of vertebrate haemoglobin
In vertebrate haemoglobin (Hb), the NH2-terminal residues of the α- and β-chain subunits are thought to play an important role in the allosteric binding of protons (Bohr effect), CO2 (as carbamino derivatives), chloride ions, and organic phosphates. Accordingly, acetylation of the α- and/or β-chain NH2-termini may have significant effects on the oxygenation properties of Hb. Here we investigate the effect of NH2-terminal acetylation by using a newly developed expression plasmid system that enables us to compare recombinantly expressed Hbs that are structurally identical except for the presence or absence of NH2-terminal acetyl groups. Experiments with native and recombinant Hbs of representative vertebrates reveal that NH2-terminal acetylation does not impair the Bohr effect, nor does it significantly diminish responsiveness to allosteric cofactors, such as chloride ions or organic phosphates. These results suggest that observed variation in the oxygenation properties of vertebrate Hbs is principally explained by amino acid divergence in the constituent globin chains rather than post-translational modifications of the globin chain NH2-termini.
more »
« less
- Award ID(s):
- 1736249
- PAR ID:
- 10283358
- Date Published:
- Journal Name:
- Biochemical Journal
- Volume:
- 477
- Issue:
- 19
- ISSN:
- 0264-6021
- Page Range / eLocation ID:
- 3839 to 3850
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
ABSTRACT 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 whether genetic variants in haemoglobin (Hb) that contribute to high-altitude adaptation in deer mice (Peromyscus maniculatus) are associated with evolved changes in the control of breathing. We created F2 inter-population hybrids of highland and lowland deer mice to test for phenotypic associations of α- and β-globin variants on a mixed genetic background. Hb genotype had expected effects on Hb–O2 affinity that were associated with differences in arterial O2 saturation in hypoxia. However, high-altitude genotypes were also associated with breathing phenotypes that should contribute to enhancing O2 uptake in hypoxia. Mice with highland α-globin exhibited a more effective breathing pattern, with highland homozygotes breathing deeper but less frequently across a range of inspired O2, and this difference was comparable to the evolved changes in breathing pattern in deer mouse populations native to high altitude. The ventilatory response to hypoxia was augmented in mice that were homozygous for highland β-globin. The association of globin variants with variation in breathing phenotypes could not be recapitulated by acute manipulation of Hb–O2 affinity, because treatment with efaproxiral (a synthetic drug that acutely reduces Hb–O2 affinity) had no effect on breathing in normoxia or hypoxia. Therefore, adaptive variation in Hb may have unexpected effects on physiology in addition to the canonical function of this protein in circulatory O2 transport.more » « less
-
Abstract A key question in evolutionary biology concerns the relative importance of different sources of adaptive genetic variation, such as de novo mutations, standing variation, and introgressive hybridization. A corollary question concerns how allelic variants derived from these different sources may influence the molecular basis of phenotypic adaptation. Here, we use a protein-engineering approach to examine the phenotypic effect of putatively adaptive hemoglobin (Hb) mutations in the high-altitude Tibetan wolf that were selectively introgressed into the Tibetan mastiff, a high-altitude dog breed that is renowned for its hypoxia tolerance. Experiments revealed that the introgressed coding variants confer an increased Hb–O2 affinity in conjunction with an enhanced Bohr effect. We also document that affinity-enhancing mutations in the β-globin gene of Tibetan wolf were originally derived via interparalog gene conversion from a tandemly linked β-globin pseudogene. Thus, affinity-enhancing mutations were introduced into the β-globin gene of Tibetan wolf via one form of intragenomic lateral transfer (ectopic gene conversion) and were subsequently introduced into the Tibetan mastiff genome via a second form of lateral transfer (introgression). Site-directed mutagenesis experiments revealed that the increased Hb–O2 affinity requires a specific two-site combination of amino acid replacements, suggesting that the molecular underpinnings of Hb adaptation in Tibetan mastiff (involving mutations that arose in a nonexpressed gene and which originally fixed in Tibetan wolf) may be qualitatively distinct from functionally similar changes in protein function that could have evolved via sequential fixation of de novo mutations during the breed’s relatively short duration of residency at high altitude.more » « less
-
The genes that encode the α- and β-chain subunits of vertebrate hemoglobin have served as a model system for elucidating general principles of gene family evolution, but little is known about patterns of evolution in amniotes other than mammals and birds. Here, we report a comparative genomic analysis of the α- and β-globin gene clusters in sauropsids (archosaurs and nonavian reptiles). The objectives were to characterize changes in the size and membership composition of the α- and β-globin gene families within and among the major sauropsid lineages, to reconstruct the evolutionary history of the sauropsid α- and β-globin genes, to resolve orthologous relationships, and to reconstruct evolutionary changes in the developmental regulation of gene expression. Our comparisons revealed contrasting patterns of evolution in the unlinked α- and β-globin gene clusters. In the α-globin gene cluster, which has remained in the ancestral chromosomal location, evolutionary changes in gene content are attributable to the differential retention of paralogous gene copies that were present in the common ancestor of tetrapods. In the β-globin gene cluster, which was translocated to a new chromosomal location, evolutionary changes in gene content are attributable to differential gene gains (via lineage-specific duplication events) and gene losses (via lineage-specific deletions and inactivations). Consequently, all major groups of amniotes possess unique repertoires of embryonic and postnatally expressed β-type globin genes that diversified independently in each lineage. These independently derived β-type globins descend from a pair of tandemly linked paralogs in the most recent common ancestor of sauropsids.more » « less
-
ABSTRACT Crocodilians are unique among vertebrates in that their hemoglobin (Hb) O2 binding is allosterically regulated by bicarbonate, which forms in red blood cells 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 purified 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 O2 saturation of Hb. We propose that both effectors bind to an unidentified positively charged site containing a reactive amino group in the low-O2 affinity T conformation of 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 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. Our results show a tight and unusual linkage between O2 and CO2 transport in the blood of crocodilians, where the build-up of erytrocytic 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.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1042/BCJ20200623
