maenas (intertidal zone) and Segonzacia mesatlantica - Station ...

176 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al.
(Table 4) contd….
Profils
A AB B
Mass (Da) of Globin Subunits
M3–15891 T1 T1 T2
M4–15930 T3 T3
Monomers forming the disulfide
bounded trimers (±2Da)
M5 – 16 018 T4 T4,6
M6 – 16 035 T5,7 T7
M7 – 16 664 T1,4 T1,3,4,6,7 T2,3,7
M8 – 16 981 T1,4 T1,4,5
M9 – 17 032 T2,3,6,7 T2,3,7
reproduce it. This condition is essential to obtain biologically
relevant data and to avoid any experimental degradation or
aggregation due to inadequate solvent. It also permits to test
the effect of changes in buffer composition on the aggregation
state, for example to test the influence of divalent
cations on complex stability and to follow dissociation and
association mechanisms. Light scattering at one angle is very
widely used to follow dissociation and reassociation of multimeric
proteins; it was an important method used by
Herskovits to investigate the dissociation of gastropods
hemocyanins [122, 130].
MS can also be used to investigate differences between
experimental conditions. Aggregation states in hydrothermal
vent crabs exposed to hypoxia and hyperoxia were monitored
by noncovalent ESI-MS [74]. Segonzacia mesatlantica
were held at deep-sea pressure under hypoxia or hyperoxia
alternatively for 6h periods. ESI-MS analysis detected hexamers,
dodecamers and 18-mers in crabs from both conditions.
Two overlapping distributions were detected for the
dodecamer and 18-mer ion series, revealing the presence of
two different forms for each aggregation state. Most interestingly,
a difference in the proportion of each dodecamer species
was detected between the two conditions (ratio 21:79
after hypoxia and 27:73 after hyperoxia). These experiments
were made on hemolymph pooled from 4 crabs and it is still
possible that random interindividual variability might exist
within the pools, but these results suggest an environmental
effect upon dodecamer proportions and in any case demonstrate
the possibility to detect precise adjustment in proportion
of very close complexes in different experimental conditions.
9.2. Investigating the Role of Phenotypic Plasticity in Response
to Environmental Challenge at the Subunit Level
As mentioned earlier, crustacean Hc present a very high
potential phenotypic plasticity due to (a) the simultaneous
existence of several aggregation states made of numerous
subunits (mainly hexamer and dodecamer) and (b) the diversity
of the subunits types. These two elements allow many
potential combinations to be produced at the molecular level,
with modulation of oxygen binding properties. Crustaceans
can live in a wide variety of habitats: some are terrestrial
species (e.g. land crab Birgus latro) and most are aquatic
species. They are found from fresh water to sea water, from
the intertidal zone to deep-sea hydrothermal vents. The comparative
study of physiological mechanisms occurring in the
very large range of conditions experienced by the different
groups is of great interest. It is also very appealing to study
some species of which individuals can support changing
conditions. Contrary to the crab Cancer pagurus, which can
only survive in non-diluted sea water, Carcinus maenas and
Callinectes sapidus can support very diluted sea water in
estuary areas and large pO2 range. Deep-sea hydrothermal
vent species living in the mixing zone between sea water and
hydrothermal fluid are exposed to rapid and unpredictable
changes [131].
The diversity of experienced environment suggests the
presence of original physiological adaptations. Many
mechanisms are at work: ventilatory and circulatory rates
compensation, metabolism adjustment [132]. Hemocyanin
oxygen affinity can be modified by hemolymph modulators
(inorganic and organic ions) [25]. Phenotypic plasticity has
been observed in several situations. It is related to ontogeny
when different subunits are expressed during successive developmental
stages as in the Dungeness crab Cancer magister
[133-135], as is observed for some vertebrates which
switch from fetal hemoglobin to adult hemoglobin. Environmental
changes induce phenotypic changes in the blue
crab Callinectes sapidus or the spiny lobster Panulirus elephas:
individuals caught from different conditions exhibit
different subunits [27, 29]. Genetic differences cannot explain
this observation since the subunit expression pattern
changes when individuals are transferred to different conditions.
It has been proved that these composition changes are
related with functional property changes in the case of
C. sapidus, P. elephas and P. mauritanicus [26-28]. An environmental
influence has also been evidenced in the case of
the Hb from the microcrustacean Daphnia magna [136].
Two approaches to comparative phenotypic plasticity are
possible. Comparison between different species living in
different environments should help to outline long-term adaptations.
On the other hand, comparison between individuals
of the same species exposed to different conditions
should help to detect short-term phenotypic adaptations.
Subunits are to be identified precisely to investigate phenotypic
plasticity involved in short-term adaptation. As mass
spectrometry is a very precise and accurate method for de-
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