healthmedicinet

Natural selection in octopus haemocyanin

Mollusc haemocyanin evolved from a ?-subclass tyrosinase some 740 million years ago
2], 33] and has evolved independently of arthropod haemocyanin since this time 34], 35]. Cephalopod haemocyanin emerged about 520 million years ago 2] with modern coleoid cephalopods (e.g. octopods, squids and sepiids) separating from
the ancient and sluggish nautiloids around 420 million years ago 2]. Since this time, the haemocyanin of coleoid cephalopods has evolved to keep pace
with the increased metabolic demands imposed by an increasingly competitive environment,
particularly since the emergence of the faster moving fishes 8]–10]. Given the several hundreds of millions of years of evolutionary history, it is thus
not surprising that the cephalopod haemocyanin gene underwent extensive purifying
selection (Fig. 4) leading to widespread sequence conservation as has been confirmed for Enteroctopus dofleini23]. High conservation of sites being directly involved in oxygen binding – such as the
copper binding centre – indicate rather indirect mechanisms modulating oxygen binding
in octopods.

Nevertheless, comparisons of two partial haemocyanin regions among octopods from various
climates revealed evidence for natural selection acting on the Octopus haemocyanin
gene. This is indicated by phylogenetic deviations between the mitochondrial genes
and the two investigated haemocyanin regions (Figs. 1, 3) and also by the presence of variable sites within otherwise highly conserved regions
(Fig. 4a), and lastly pinpointed by sites with increased rates of non-synonymous substitutions
confirmed by various selection tests (Fig. 4b, c, Table 1). Unfortunately, studies assessing positive selection in mollusc haemocyanins are
lacking and at least scarce in other groups. Positive selection was reported for lobster
haemocyanins due to high non-synonymous substitution rates estimated for whole sequence
regions in some species 36], 37], however analysis of single codon sites failed to confirm these findings 37]. This first report of positive selection in cephalopod haemocyanin indicates the
presence of sites potentially involved in functional adaptation of haemocyanin.

Structural links to protein function

Although Octopus haemocyanin contains sites identified to be positively selected one needs to assess
their functional relevance. We thus analysed all 13 selected sites for their structural
properties to deduce potential functional effects.

Our analyses showed that selection prevailed on residues located at the surface of
the haemocyanin FU g and FU f, mostly affecting polar/charged properties (Fig. 5, Table 1), suggesting functional relevance for tertiary or quaternary interactions. Polar
and particularly charged surface residues play a major role in stabilizing contacts
among functional units of cephalopod haemocyanin via salt bridges 21]. Further, cooperativity, often found to be pronounced in cephalopods 38], has been proposed to operate via various interfaces among all seven FU types 21]. The partial regions analysed in this study are part of FU f and FU g, which interact
with other FU via various surface interfaces. These comprise the closely spaced morphological
unit interface FU c?f, the horizontal tier interface FU e?f and the arc wall interface
FU g1/g2?d as well as the more distant major groove interface FU a?f and the arc morphological
unit interface FU g1?g2 characterized by weaker interactions 21]. Based on the detailed interface models of Nautilus haemocyanin 21], contact residues for the morphological unit interface FU c?f were identified as
F2393, 2428WHFDRT2433 and P2479 for the octopod haemocyanin sequences analysed in
this study (corresponds to H2401, 2437WKYDRL2442 and H2488 in Nautilus). Interestingly, this region did not contain any of the variable and positively selected
sites, and in fact, was highly conserved across all 113 sequences (Fig. 4a, Additional file 4). Therefore, the morphological unit interface FU c?f seems a less likely target for
direct functional regulation, although significant sequence differences of haemocyanins
between octopods and Nautilus suggest further or different contact regions at this interface than proposed for
Nautilus haemocyanin only. Contact residues for the alternative interfaces FU e?f , FU g1/g2?d,
FU a?f and FU g1?g2 were located outside the analysed sequence region and remain to
be assessed in subsequent studies.

Only three buried residues were identified to be positively selected, out of which
residue 2545 has high potential to be an allosteric site. This is due to its linking
position between the copper binding histidine (His2543) and the nearby FU surface
as well as its immediate neighbourhood composed of hydrophobic and hydrophilic amino
acids. These create a hydrophobic/hydrophilic contrast, which promotes metal binding,
particularly in the presence of the sulphur carrying methionine 39]–41]. A conformational movement of residue 2545 upon allosteric binding could easily transfer
to the two amino acid distant copper binding His2543 and affect oxygen binding, as
minor shifts of only 0.7 Å between the coordinated copper ions suffice to change oxygenation
21].

The remaining positively selected buried residues 2575 and 2442 are less likely to
be involved in functional regulation. Despite the proximity of site 2575 to the copper
binding His2571, neither alanine, valine nor threonine had the possibility to interact
directly with the active site. Alanine and valine are very non-reactive 42] and were too distant to affect the active site. Direct polar interaction of threonine
was also unlikely, as the only nearby polar group, the terminal amino group of His2571,
was involved in a covalent peptide bond. Moreover, inward facing residues involved
at site 2442 were exclusively hydrophobic (methionine, isoleucine, leucine or valine),
non-reactive and due to their large distance to the active site unlikely to be involved
in functional regulation.

Although variation and positive selection were most significant at site 2503 (Fig. 4), functional relevance is rather unlikely. Site 2503 is located at the beginning
of the linker region connecting FU f and FU g1/g2 that, like all inter-FU linkers,
comprise a long (34–57 Å), drawn-out sequence between 12–20 amino acids 21]. Given this high length and the capacity to extend further, as linkers are unlikely
to extend to their maximum length 21], there is little chance that the identified substitutions among alanine, valine,
threonine, or serine (Additional file 4: Figure S4) affect the positioning or conformation of the ca. 51 Å distant FU g1/g2.

Correlates of temperature adaptation

Temperature affects oxygen supply 13], a challenge that octopods have been required to overcome in order to colonize oceans
from the tropics to the poles, where they now thrive at high diversity and abundance
4]. Low temperatures increase the affinity of haemocyanin for oxygen 38], 43], thus hampering oxygen release to the tissue, to an extent that it limits oxygen
supply in cephalopods 15], 16]. On the other hand, lower temperatures increase levels of physically dissolved oxygen
in the blood/haemolymph 44], 45] and decrease mass specific metabolic demands for oxygen of ectothermic animals 46]–48], which enabled a reduction or even a complete loss of haemoglobin in Antarctic notothenioid
fishes 45], 49]. Antarctic octopods similarly benefit from relaxed oxygen requirements in the cold
(e.g. metabolic rate of 0.319 mmol O
₂
kg
^?1
(wet mass) h
^?1
at 0 °C in the Antarctic Pareledone charcoti vs. 2.672 mmol O
₂
kg
^?1
at 21 °C in the subtropical Octopus vulgaris48], 50] and dissolved oxygen contributing 18.5 % to total haemolymph oxygen content in Pareledone charcoti16]). Nevertheless, Antarctic octopods continue to rely on active oxygen transport. This
is marked by 39 % to 46 % higher concentrations of haemocyanin in the haemolymph and
functional modifications of haemocyanin comprising a lower affinity for oxygen, increased
rates of pH dependent oxygen release as well as a shift of the pH sensitive range
of oxygen binding towards higher pH values compared to octopods from warmer waters
16].

So far, our data has highlighted positive selection acting on surface charges and
a potential allosteric site. We thus assessed whether this explains differences among
octopods from polar, temperate, subtropical or tropical climates and in particular
the functional adaptations observed in cold-adapted Antarctic octopods 16]. The analysis of polar surface residues revealed a clear distinction between cold
and warm adapted octopods dominated by a decrease of glutamic acid and an increase
of positively charged amino acids towards colder climates, causing a higher net surface
charge in polar octopods compared to octopods from warmer waters (Fig. 6). Importantly, the comparison between the Antarctic octopus Pareledone charcoti and the temperate Enteroctopus dofleini revealed that this pattern not only applies to the partial regions FU f-g and FU
g but also to most of the entire haemocyanin gene (except FU a, Fig. 7). This indicates that climate dependent selection of surface charge properties occurred
in parallel for several FUs, although full haemocyanin sequences of more and particularly
tropical species need to be included to further substantiate these findings.

Changes of net surface charge properties help to explain how cold-adapted octopods
attenuate the detrimental effect of increased oxygen affinity. Perutz 51] provided the first structural explanation for altered oxygen affinity in haemoglobin,
based on a His-Asp salt bridge linking two haemoglobin subunits. High ambient pH deprotonates
the histidine and disrupt this salt bridge, which destabilizes or dilates the haemoglobin
quaternary structure and thus increases oxygen affinity by adjacent subunits. Haemocyanin
may respond similarly. It not only dissociates into its subunits at higher pH 52], 53] but is also highly pH sensitive with Bohr coefficients well below ?1 26], 54]. Further, unlike intracellular haemoglobin 55], haemocyanin is not protected from changes of haemolymph pH. Consequently, one would
expect even larger effects of pH disturbances on haemocyanin structure than on haemoglobin.
Octopods living in polar waters face higher haemolymph pH than warm water species
due to the temperature dependency of equilibrium constants (pK) of ionisable groups,
particularly the imidazole groups of proteins (i.e. alpha stat pattern, Fig. 8a, 16], 56], 57]). Such high haemolymph pH contributes to increased oxygen affinity and to the observed
impairment of venous oxygen release towards colder temperatures 15], 16]. As a result, an increase of oxygen affinity in response to a cold-induced pH increase
may be reduced or prevented if haemocyanin quaternary structure remains stable despite
higher pH. The disruption of salt bridges linking haemocyanin FUs upon pH changes,
as they occur during temperature changes, depends on the pK values of their ionisable
groups, which are not fixed but variable depending on their protein environment 58]. Charge-charge interactions at the protein surface are among the most important factors
disturbing the pK of ionisable groups, as evidenced by increased numbers of positively
charged lysine, which raise the pK value of several surface residues, by up to 2.19
units 58]. Experimental substitutions from glutamic acid to lysine also confirmed increased
stability (1.1 kcal mol
^?1
) due to charge-charge interactions at the surface of Ribonuclease T1 59]. Therefore, considering a more positive net surface charge in polar octopods, due
to increased numbers of positively charged amino acids and decreased numbers of the
negatively charged glutamic acid (Fig. 6), charge-charge interactions are likely to raise pK of residues linking FU interfaces.
Consequently, salt bridges withstand disturbance by high pH occurring at low temperatures
and retain the stability of the haemocyanin quaternary structure and accordingly the
affinity for oxygen. This is in good agreement with oxygen binding in the Antarctic
octopod Pareledone charcoti, which showed a lower oxygen affinity than the subtropical octopod Eledone moschata at the same pH and temperature (10 °C 16]). This compensation of oxygen affinity was particularly due to a shift of the pH
dependent oxygen binding range in Pareledone charcoti towards higher pH (Fig. 8b, 16]). Theoretical buffer lines of surface residues of the partial haemocyanin sequence
FU f-g confirm the more positive net surface charge in Pareledone charcoti compared to Eledone moschata at a venous pH of 7.27 (Fig. 8c). Altered surface charge properties were also suggested to facilitate oxygen secretion
of haemoglobin (i.e. Bohr and Root effect) via a decrease of non-conserved histidine
residues, which lower the capacity to buffer pH 60]. Similarly, myoglobin underwent changes of net surface charge to increase oxygen
storage capacity in diving mammals via increased electrostatic repulsion, which reduces
self-association of myoglobin 61]. However, unlike gastropod haemocyanin 22], 62], cephalopod haemocyanin does not assemble to multi-decameric complexes 63]. Thus electrostatic interactions likely occur intrinsically among the haemocyanin
FUs.

Fig. 8. Functional and structural pH dependence. a pH change of octopod haemolymph following an imidazole like alpha stat pattern of
?0.0153 pH units / °C (redrawn from 16]) b) Differences between the Antarctic Pareledone charcoti (blue) and the Mediterranean Eledone moschata (red) regarding pH dependent oxygen binding at 10 °C and 1kPa PO
₂
(shaded area denotes 95 % C.I., ?=?5, data taken from 16]) and c) net surface charge of the partial haemocyanin region FU f-g at pH 7.27 based on
six partial haemocyanin sequences for each species, whose buffer lines partly overlap.
The dashed vertical line indicates the venous pH at 10 °C interpolated from a)

Further, the proposed charge-charge interactions at the surface of FU g and FU f modulate
oxygen binding rather via indirect modifications. This is underpinned by the high
conservation of the morphological unit interface FU c?f and the more uniform distribution
of positively selected residues at the protein surface.

The presence of two very distinct isoforms in FU g, which does not follow the observed
correlation between net surface charge and climatic origin, also supports the view
of functionally divergent isoforms that are regulated via differential expression
to enable flexible responses to changing environmental or metabolic conditions 64], 65]. Indeed, native page gel electrophoresis showed differential expression of haemocyanin
isoforms in the Antarctic squid Moroteuthis ingens (Oellermann et al. unpublished). However, this could not be documented for octopod
haemocyanin so far as their isoforms rarely separate on native gels, except for the
Antarctic octopod Pareledone turqueti, which showed a relatively even expression between two distinct haemocyanin isoforms
(Oellermann et al. unpublished). Similarly, in the cuttlefish Sepia officinalis isoforms levels of haemocyanin mRNA do not differ in adult specimens or in response
to temperature or hypercapnia and change only during ontogeny 25]. Therefore it remains unclear if octopods employ differential haemocyanin expression
to modulate oxygen binding and if polar octopods express their “high net surface charge”
isoform of FU g (Fig. 6b) at significant levels to modulate oxygen binding.

Distinct evolutionary histories of the two Antarctic octopod families may explain
why the Enteroctopodidae did not share the same pattern of charged surface residues
as the Megaleledonidae (Fig. 6). The Megaleledonidae are thought to have had their evolutionary origins in Antarctica
before the onset of glaciation around 50 Ma years ago 66] whereas the Enteroctopodidae are thought to have had their evolutionary origins in
shallow Northern Hemisphere waters from where they moved in to the deep sea and subsequently
to Antarctic waters 27], 67]. Therefore, Enteroctopodidae may have followed a diverging strategy to cope with
cold temperatures either marked by a total lack of compensation or via physiological
adjustments that did not involve haemocyanin. Such alternative adjustments may comprise
increased blood circulation or ventilation, as indicated in Benthoctopus spp. (Enteroctopodidae) that shows higher metabolic enzyme activities in mantle muscle
than Graneledone boreopacificus (Megaleledonidae), which shares the same deep sea habitat 68].

Moreover, residue 2545 has the potential as an allosteric site and interestingly contained
mostly methionine in polar octopods and mostly leucine in tropical octopods. The sulphur
group of methionine plays an important role in coordinating cations such as copper
ions in other “blue” proteins such as plastocyanins or azurins 40] as well as in copper trafficking protein 69], 70]. In fungal laccases, substitution of methionine with non-coordinating leucine or
phenylalanine breaks a copper-methionine interaction, likely leading to significant
changes of the redox potential at this site 40], 71]. Experimental substitution of methionine with norleucine further confirmed specific
binding of copper by the sulphur group of methionine 72]. Although methionine was most frequently found to bind copper, it is also able to
form stable complexes with magnesium 41], indicating a general affinity for divalent cations. Therefore, a hydrophilic/hydrophobic
contrast 39] together with a selective preference of methionine in polar and leucine in warm water
octopods at site 2545 may serve to regulate oxygen affinity in response to temperature
adaptation via altered affinity to allosteric metals. Such intrinsic regulation would
provide a reasonable mechanism to exploit for example magnesium as oxygen affinity
regulator despite the octopods’ inability to regulate extracellular magnesium concentrations
16].