Crocodilian
hemoglobin: Experiments on Ancient Proteins Find Mutations more Numerous and Nuanced
than Once Thought
From: University of Nebraska-Lincoln
January 12, 2023 -- The
crocodilian edition of hemoglobins -- the scuba tanks of the blood -- work so
well that crocs can go hours without air. The hyper-efficiency of that
adaptation has led some biologists to wonder why, of all the jawed vertebrates,
crocodilians were the lone group to hit on such an optimal solution to making
the most of a breath. After resurrecting the hemoglobin of ancient crocodilian
ancestors, a team may have an answer.
It can pogo-stick along
at 50-plus miles per hour, leaping 30-odd feet in a single bound. But that
platinum-medal athleticism falls by the wayside at a sub-Saharan riverside, the
source of life and death for the skittish impala stilling itself for a drink in
100-degree heat. A Nile crocodile has silently baptized itself in that same
muddy river for the past hour. When the unseen apex predator lashes from the
water to seize the impala, its infamous teeth latch onto a hindquarter, jaws
clenching with 5,000 pounds of force. Yet it's the water itself that does the
killing, with the deep-breathed reptile dragging its prey to the deep end to
drown.
The success of the
croc's ambush lies in the nanoscopic scuba tanks -- hemoglobins -- that course
through its bloodstream, unloading oxygen from lungs to tissues at a slow but
steady clip that allows it to go hours without air. The hyper-efficiency of that
specialized hemoglobin has led some biologists to wonder why, of all the jawed
vertebrates in all the world, crocodilians were the lone group to hit on such
an optimal solution to making the most of a breath.
By statistically
reconstructing and experimentally resurrecting the hemoglobin of an archosaur,
the 240-million-year-old ancestor of all crocodilians and birds, the University
of Nebraska-Lincoln's Jay Storz and colleagues have gleaned new insights into
that why. Rather than requiring just a few key mutations, as earlier research
suggested, the unique properties of crocodilian hemoglobin stemmed from 21
interconnected mutations that litter the intricate component of red blood
cells.
That complexity, and
the multiple knock-on effects that any one mutation can induce in hemoglobin,
may have forged an evolutionary path so labyrinthine that nature failed to
retrace it even over tens of millions of years, the researchers said.
"If it was such an
easy trick -- if it was that easy to do, just making a few changes -- everyone
would be doing it," said Storz, a senior author of the study and Willa
Cather Professor of biological sciences at Nebraska.
All hemoglobin binds
with oxygen in the lungs before swimming the bloodstream and eventually
releasing that oxygen to the tissues that depend on it. In most vertebrates,
hemoglobin's affinity for capturing and holding oxygen is dictated largely by
molecules known as organic phosphates, which, by attaching themselves to the
hemoglobin, can coax it into releasing its precious cargo.
But in crocodilians --
crocodiles, alligators and their kin -- the role of organic phosphates was
supplanted by a molecule, bicarbonate, that is produced from the breakdown of
carbon dioxide. Because hardworking tissues produce lots of carbon dioxide,
they also indirectly generate lots of bicarbonate, which in turn encourages
hemoglobin to dispense its oxygen to the tissues most in need of it.
"It's a
super-efficient system that provides a kind of slow-release mechanism that
allows crocodilians to efficiently exploit their onboard oxygen stores,"
Storz said. "It's part of the reason they're able to stay underwater for
so long."
As postdoctoral
researchers in Storz's lab, Chandrasekhar Natarajan, Tony Signore and Naim
Bautista had already helped decipher the workings of the crocodilian
hemoglobin. Alongside colleagues from Denmark, Canada, the United States and
Japan, Storz's team decided to embark on a multidisciplinary study of how the
oxygen-ferrying marvel came to be.
Prior efforts to
understand its evolution involved incorporating known mutations into human
hemoglobin and looking for any functional changes, which were usually scant.
Recent findings from his own lab had convinced Storz that the approach was
flawed. There were plenty of differences, after all, between human hemoglobin
and that of the ancient reptilian creatures from which modern-day crocodilians
evolved.
"What's important
is to understand the effects of mutations on the genetic background in which
they actually evolved, which means making vertical comparisons between
ancestral and descendant proteins, rather than horizontal comparisons between
proteins of contemporary species," Storz said. "By using that
approach, you can figure out what actually happened."
So, with the help of
biochemical principles and statistics, the team set out to reconstruct
hemoglobin blueprints from three sources: the 240-million-year-old archosaur
ancestor; the last common ancestor of all birds; and the 80-million-year-old
shared ancestor of contemporary crocodilians. After putting all three of the
resurrected hemoglobins through their paces in the lab, the team confirmed that
only the hemoglobin of the direct crocodilian ancestor lacked phosphate binding
and boasted bicarbonate sensitivity.
Comparing the hemoglobin
blueprints of the archosaur and crocodilian ancestors also helped identify
changes in amino acids -- essentially the joints of the hemoglobin skeleton --
that may have proved important. To test those mutations, Storz and his
colleagues began introducing certain croc-specific mutations into the ancestral
archosaur hemoglobin. By identifying the mutations that made archosaur
hemoglobin behave more like that of a modern-day crocodilian, the team pieced
together the changes responsible for those unique, croc-specific properties.
Counter to conventional
wisdom, Storz and his colleagues discovered that evolved changes in
hemoglobin's responsiveness to bicarbonate and phosphates were driven by
different sets of mutations, so that the gain of one mechanism was not
dependent on the loss of the other. Their comparison also revealed that, though
a few mutations were enough to subtract the phosphate-binding sites, multiple
others were needed to eliminate phosphate sensitivity all together. In much the
same way, two mutations seemed to directly drive the emergence of bicarbonate
sensitivity -- but only when combined with or preceded by other, easy-to-miss
mutations in remote regions of the hemoglobin.
Storz said the findings
speak to the fact that a combination of mutations might yield functional
changes that transcend the sum of their individual effects. A mutation that
produces no functional effect on its own might, in any number of ways, open a
path to other mutations with clear, direct consequences. In the same vein, he
said, those later mutations might influence little without the proper
stage-setting predecessors already in place. And all of those factors can be
supercharged or waylaid by the environment in which they unfold.
"When you have
these complex interactions, it suggests that certain evolutionary solutions are
only accessible from certain ancestral starting points," Storz said.
"With the ancestral archosaur hemoglobin, you have a genetic background
that makes it possible to evolve the unique properties that we see in
hemoglobins of modern-day crocodilians. By contrast, with the ancestor of
mammals as a starting point, it may be that there's some way that you could
evolve the same property, but it would have to be through a completely
different molecular mechanism, because you're working within a completely
different structural context."
For better or worse,
Storz said, the study also helps explain the difficulty of engineering a human
hemoglobin that can mimic and approach the performance of the crocodilian.
"We can't just
say, 'OK, it's mainly due to these five mutations. If we take human hemoglobin
and just introduce those mutations, voilà , we'll have one with those same exact
properties, and we'll be able to stay underwater for two hours, too,'"
Storz said. "It turns out that's not the case.
"There are lots of
can't-get-there-from-here problems in the tree of life."
Storz, Natarajan and
Bautista conducted the study with Signore, now at the University of Manitoba;
Aarhus University's Angela Fago; Mississippi State University's Federico
Hoffmann; and Yokohama City University's Jeremy Tame. The researchers detailed
their findings in the journal Current Biology, receiving support
from the National Science Foundation and National Institutes of Health.
https://www.sciencedaily.com/releases/2023/01/230112134747.htm