Evolution of an extreme hemoglobin phenotype contributed to the sub-Arctic specialization of extinct Steller’s sea cows
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eLife assessment
This important study functionally characterizes hemoglobin from Steller's sea cow, a cold-water adapted sirenian that went extinct ~250 years ago. Using ancestral sequence reconstruction, site-directed mutagenesis and biochemical assays to compare Steller's hemoglobin to those from (sub)tropical extant sea cows (all of which are proficient divers despite lacking massive muscle oxygen storage), the authors build a solid case for the molecular basis of cold adaptation, centered around an increased solubility and higher oxygen carrying capacity. Remarkably, a single amino acid replacement would explain most of the distinctive functional features of this hemoglobin, which include a hitherto unknown resistance to DPG. Overall, this work will be of interest to evolutionary biologists, physiologists, and biochemists, as well as an enjoyable and informative read for the general public.
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Abstract
The extinct Steller’s sea cow ( Hydrodamalis gigas ; †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; α 2 β/δ 2 ) of this sirenian is shown to harbor a fixed amino acid replacement at an otherwise invariant position (β/δ82Lys→Asn) that alters multiple aspects of Hb function. First, our functional characterization of recombinant sirenian Hb proteins demonstrates that the Hb-O 2 affinity of this sub-Arctic species was less affected by temperature than those of living (sub)tropical sea cows. This phenotype presumably safeguarded O 2 delivery to cool peripheral tissues and largely arises from a reduced intrinsic temperature sensitivity of the H. gigas protein. Additional experiments on H. gigas β/δ82Asn→Lys mutant Hb further reveal this exchange renders Steller’s 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, β/δ82Lys→Asn moreover underlies the secondary evolution of a reduced blood-O 2 affinity phenotype that would have promoted heightened tissue and maternal/fetal O 2 delivery. This conclusion is bolstered by analyses of two Steller’s sea cow prenatal Hb proteins (Hb Gower I; ζ 2 ε 2 and HbF; α 2 γ 2 ) that suggest an exclusive embryonic stage expression pattern, and reveal uncommon replacements in H. gigas HbF (γ38Thr→Ile and γ101Glu→Asp) that increased Hb-O 2 affinity relative to dugong HbF. Finally, the β/δ82Lys→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.
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Author Response
Reviewer #1 (Public Review):
In this study the authors sought to address the issue of whether the Steller's sea cow -- a massive extinct sirenian ("sea cow") species that differs from its living relatives (manatees and dugongs) not only in body mass but also in having inhabited cold climates in the northern Pacific -- had hemoglobin adaptations that enhanced the species' thermoregulatory capacities relative to those of the extant species, which are restricted to relatively warm waters. To do so, the authors synthesized recombinant hemoglobin proteins of all the major sea cow lineages and used these data to assess differences in O2 binding, Hb solubility, responses to allosteric effectors, and thermal sensitivity. The work presented is very innovative and in my opinion convincingly demonstrates that the Steller's sea …
Author Response
Reviewer #1 (Public Review):
In this study the authors sought to address the issue of whether the Steller's sea cow -- a massive extinct sirenian ("sea cow") species that differs from its living relatives (manatees and dugongs) not only in body mass but also in having inhabited cold climates in the northern Pacific -- had hemoglobin adaptations that enhanced the species' thermoregulatory capacities relative to those of the extant species, which are restricted to relatively warm waters. To do so, the authors synthesized recombinant hemoglobin proteins of all the major sea cow lineages and used these data to assess differences in O2 binding, Hb solubility, responses to allosteric effectors, and thermal sensitivity. The work presented is very innovative and in my opinion convincingly demonstrates that the Steller's sea cow had remarkable hemoglobin adaptations that allowed for an extreme range extension into cool waters despite several physiological constraints that are inherent to the sirenian (and paenungulate, afrotherian, etc.) clade. I did not detect any obvious weaknesses of the paper, whereas the use of ancient DNA to resurrect 'extinct' hemoglobins, and the various analyses of these extinct hemoglobins alongside those of extant relatives is very exciting and are major strengths of the paper that make this study a very important advance for our understanding of Steller's sea cow's paleophysiology, as well as our understanding of the potential for extreme hemoglobin phenotypes that have not been documented among living species. Moving forward, these methods can be used to study aspects of the paleophysiology of other recently extinct mammals. I applaud the authors on an excellent and innovative study that significantly augments our understanding of the Steller's sea cow.
We sincerely appreciate the constructive comments of this reviewer.
Reviewer #2 (Public Review):
This manuscript is an impressive "resurrection" of physiology regarding an enigmatic though unfortunately extinct species, and their potential adaptation to cold-water environments. I am largely convinced of their findings, which I feel are very straightforward and thorough.
One place where the authors perhaps fell a bit short was regarding some conclusions associated with maternal/fetal oxygen delivery. The sirenian versions of fetal & embryonic hemoglobin genes have been identified and assessed to some degree in previously published work the same research group. I feel the manuscript would have benefited from actual analysis of the fetal & embryonic hemoglobin (epsilon, gamma, zeta) to strengthen their assertions.
Again, we appreciate the kind words and valid concern of this reviewer regarding a potential shortcoming of the maternal/fetal gas exchange discussion. As noted above, we previously collected physiological data from two pre-natal Steller’s sea cow Hb isoforms that were initially intended to form a stand-alone publication on this topic. However, we have elected to include this data here to better support our claims and provide a more complete picture of maternal/fetal oxygen delivery in this extinct species.
Reviewer #3 (Public Review):
Signore et al. synthesized and functionally characterized the recombinant adult hemoglobin (Hb) proteins of extant, extinct, and ancestral sirenians to explore the putative role of Hb in helping Steller's sea cows adapt to life in extremely cold waters. The functional comparisons show that the Hb of the subarctic Steller's sea cows differs in multiple biochemical properties relative to the Hbs of the two extant sirenians in the study, the Florida manatee, and the dugong and also from the Hb inferred for the common ancestor of Steller's sea cow and dugong. Specifically, the Steller's sea cow shows reduced oxygen binding affinity, reduced sensitivity to the allosteric cofactors DPG, Cl-, and H+, increased solubility, and reduced thermal sensitivity. DPG plays an important role in regulating Hb oxygen affinity in mammals, and the lack of sensitivity to it is unique to the Hb of Steller's sea cow. Sequence comparisons show that the Hb of the Steller's sea cow differs at 11 amino acids from that of its sister group, the dugong, one of which is intriguing because it occurs in a position that is invariable among mammals at a site that is critical for DPG binding, a change from Lys to Asn in position 82 of the mature β/δ globin chain. To test the significance of this change, the authors use site directed mutagenesis to insert back a Lys in the Steller's sea cow Hb background (β/δ82Asn→Lys) and test its biochemical properties. The functional assays with the β/δ82Asn→Lys mutant indicate that reverting this position to its ancestral state drastically altered the biochemical properties of the Steller's sea cow Hb, making it functionally similar to the Hbs of manatee, dugong, and the Hb inferred for the common ancestor of Steller's sea cow and dugong.
The study's strength lies in comparing the different recombinant Hbs in an explicit evolutionary framework. The conclusions are supported by the analyses, and the results are relevant in the fields of evolutionary biology, physiology, and biochemistry because they suggest that a single amino acid substitution in a protein can have profound biochemical consequences that impact whole organism physiology.
We concur with the excellent synopsis of this reviewer. The finding that most of the functional differences between Steller’s sea cow and other sirenian Hbs can be attributed to a single amino acid replacement mirrors earlier sentiments of hemoglobin adaptation by pioneers in the field (e.g. Max Perutz). By contrast, more recent studies highlight the importance of multiple causative replacements of smaller effect and the significance of genetic background in hemoglobin evolution/adaptation (which is also evident for Steller’s sea cow Hb). We hope that the present work helps to bridge these two important evolutionary forces.
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eLife assessment
This important study functionally characterizes hemoglobin from Steller's sea cow, a cold-water adapted sirenian that went extinct ~250 years ago. Using ancestral sequence reconstruction, site-directed mutagenesis and biochemical assays to compare Steller's hemoglobin to those from (sub)tropical extant sea cows (all of which are proficient divers despite lacking massive muscle oxygen storage), the authors build a solid case for the molecular basis of cold adaptation, centered around an increased solubility and higher oxygen carrying capacity. Remarkably, a single amino acid replacement would explain most of the distinctive functional features of this hemoglobin, which include a hitherto unknown resistance to DPG. Overall, this work will be of interest to evolutionary biologists, physiologists, and biochemists, as well …
eLife assessment
This important study functionally characterizes hemoglobin from Steller's sea cow, a cold-water adapted sirenian that went extinct ~250 years ago. Using ancestral sequence reconstruction, site-directed mutagenesis and biochemical assays to compare Steller's hemoglobin to those from (sub)tropical extant sea cows (all of which are proficient divers despite lacking massive muscle oxygen storage), the authors build a solid case for the molecular basis of cold adaptation, centered around an increased solubility and higher oxygen carrying capacity. Remarkably, a single amino acid replacement would explain most of the distinctive functional features of this hemoglobin, which include a hitherto unknown resistance to DPG. Overall, this work will be of interest to evolutionary biologists, physiologists, and biochemists, as well as an enjoyable and informative read for the general public.
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Reviewer #1 (Public Review):
In this study the authors sought to address the issue of whether the Steller's sea cow -- a massive extinct sirenian ("sea cow") species that differs from its living relatives (manatees and dugongs) not only in body mass but also in having inhabited cold climates in the northern Pacific -- had hemoglobin adaptations that enhanced the species' thermoregulatory capacities relative to those of the extant species, which are restricted to relatively warm waters. To do so, the authors synthesized recombinant hemoglobin proteins of all the major sea cow lineages and used these data to assess differences in O2 binding, Hb solubility, responses to allosteric effectors, and thermal sensitivity. The work presented is very innovative and in my opinion convincingly demonstrates that the Steller's sea cow had remarkable …
Reviewer #1 (Public Review):
In this study the authors sought to address the issue of whether the Steller's sea cow -- a massive extinct sirenian ("sea cow") species that differs from its living relatives (manatees and dugongs) not only in body mass but also in having inhabited cold climates in the northern Pacific -- had hemoglobin adaptations that enhanced the species' thermoregulatory capacities relative to those of the extant species, which are restricted to relatively warm waters. To do so, the authors synthesized recombinant hemoglobin proteins of all the major sea cow lineages and used these data to assess differences in O2 binding, Hb solubility, responses to allosteric effectors, and thermal sensitivity. The work presented is very innovative and in my opinion convincingly demonstrates that the Steller's sea cow had remarkable hemoglobin adaptations that allowed for an extreme range extension into cool waters despite several physiological constraints that are inherent to the sirenian (and paenungulate, afrotherian, etc.) clade. I did not detect any obvious weaknesses of the paper, whereas the use of ancient DNA to resurrect 'extinct' hemoglobins, and the various analyses of these extinct hemoglobins alongside those of extant relatives is very exciting and are major strengths of the paper that make this study a very important advance for our understanding of Steller's sea cow's paleophysiology, as well as our understanding of the potential for extreme hemoglobin phenotypes that have not been documented among living species. Moving forward, these methods can be used to study aspects of the paleophysiology of other recently extinct mammals. I applaud the authors on an excellent and innovative study that significantly augments our understanding of the Steller's sea cow.
-
Reviewer #2 (Public Review):
This manuscript is an impressive "resurrection" of physiology regarding an enigmatic though unfortunately extinct species, and their potential adaptation to cold-water environments. I am largely convinced of their findings, which I feel are very straightforward and thorough.
One place where the authors perhaps fell a bit short was regarding some conclusions associated with maternal/fetal oxygen delivery. The sirenian versions of fetal & embryonic hemoglobin genes have been identified and assessed to some degree in previously published work the same research group. I feel the manuscript would have benefited from actual analysis of the fetal & embryonic hemoglobin (epsilon, gamma, zeta) to strengthen their assertions.
-
Reviewer #3 (Public Review):
Signore et al. the synthesized and functionally characterized the recombinant adult hemoglobin (Hb) proteins of extant, extinct, and ancestral sirenians to explore the putative role of Hb in helping Steller's sea cows adapt to life in extremely cold waters. The functional comparisons show that the Hb of the subarctic Steller's sea cows differs in multiple biochemical properties relative to the Hbs of the two extant sirenians in the study, the Florida manatee, and the dugong and also from the Hb inferred for the common ancestor of Steller's sea cow and dugong. Specifically, the Steller's sea cow shows reduced oxygen binding affinity, reduced sensitivity to the allosteric co-factors DPG, Cl-, and H+, increased solubility, and reduced thermal sensitivity. DPG plays an important role in regulating Hb oxygen …
Reviewer #3 (Public Review):
Signore et al. the synthesized and functionally characterized the recombinant adult hemoglobin (Hb) proteins of extant, extinct, and ancestral sirenians to explore the putative role of Hb in helping Steller's sea cows adapt to life in extremely cold waters. The functional comparisons show that the Hb of the subarctic Steller's sea cows differs in multiple biochemical properties relative to the Hbs of the two extant sirenians in the study, the Florida manatee, and the dugong and also from the Hb inferred for the common ancestor of Steller's sea cow and dugong. Specifically, the Steller's sea cow shows reduced oxygen binding affinity, reduced sensitivity to the allosteric co-factors DPG, Cl-, and H+, increased solubility, and reduced thermal sensitivity. DPG plays an important role in regulating Hb oxygen affinity in mammals, and the lack of sensitivity to it is unique to the Hb of Steller's sea cow. Sequence comparisons show that the Hb of the Steller's sea cow differs at 11 amino acids from that of its sister group, the dugong, one of which is intriguing because it occurs in a position that is invariable among mammals at a site that is critical for DPG binding, a change from Lys to Ans in position 82 of the mature β/δ globin chain. To test the significance of this change, the authors use site directed mutagenesis to insert back a Lys in the Steller's sea cow Hb background (β/δ82Asn→Lys) and test its biochemical properties. The functional assays with the β/δ82Asn→Lys mutant indicate that reverting this position to its ancestral state drastically altered the biochemical properties of the Steller's sea cow Hb, making it functionally similar to the Hbs of manatee, dugong, and the Hb inferred for the common ancestor of Steller's sea cow and dugong.
The study's strength lies in comparing the different recombinant Hbs in an explicit evolutionary framework. The conclusions are supported by the analyses, and the results are relevant in the fields of evolutionary biology, physiology, and biochemistry because they suggest that a single amino acid substitution in a protein can have profound biochemical consequences that impact whole organism physiology.
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