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

174 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al.
mentation methods to separate 24S and 16S species (corresponding
to dodecamer and hexamer, respectively). Hexamer
is the dominant form in isopods, euphausiids (krill), carideans
(shrimps), penaeids (prawns), most palinurids (spiny
lobsters) and Uca species (brachyuran). Dodecamer is dominant
in brachyurans (except Uca), astacids (lobsters, crayfishes),
anomurans (hermit crabs) and some Scyllarus species
(palinurid). Thalassinid shrimps (ghost shrimps) are
original since they present a 24-mer aggregation state [106].
MALLS was used to determine Hc aggregation states for
the hydrothermal shrimp Rimicaris exoculata (caridean)
[105]. A dominant hexameric form was observed with a
mass of 469 ± 9 kDa, along with minor monomeric and dodecameric
forms. This is consistent with the data previously
observed in other caridean species from shallow water.
MALLS determined masses of 950 000 kDa (12-mer) and
1 800 000 kDa (24-mer) were obtained for Penaeus monodon,
with a dominant 6-mer (mass undetermined) and a minor
24-mer [123]. Non-covalent ESI-MS was also used to
determine the complex masses for the brachyurans Bythograea
thermydron, Segonzacia mesatlantica and Carcinus
maenas (Table 3). Dodecamers and hexamers were detected
in agreement with previous observations, and their mass
could be precisely determined. Higher aggregation states
were also discovered in these crabs [62].
Subunit diversity in a given species was investigated in
several groups [29, 106, 118, 124-127]. These works used
native PAGE and evidenced high heterogeneity among species
but no clear relation was found between native PAGE
pattern and taxonomic position. However, Hc subunit heterogeneity
revealed as a useful tool for asserting cryptic or
morphologically similar species [118, 125]. An hypothesis is
that Hc could be among the first protein to diverge when a
speciation event occurs [118, 126].
Denaturing ESI-MS experiments allowed determination
of subunit masses for caridean and brachyuran species (Table
3). This method showed higher subunit diversity than
could be previously detected by PAGE method. Application
of these methods to comparison of Hc diversity within other
decapod groups should be of interest since subunits could be
identified precisely and unambiguously and precise mass of
complexes could be determined. Since no relation was observed
between PAGE pattern and taxonomy, it is possible
that no other relation could be observed even with the high
resolution of MS. Even in this case, MS would indubitably
be of great interest for distinguishing between sibling species
or even for populations within a unique species.
8. FROM STRUCTURE TO BIOLOGICAL POPULA-
TIONS: HB POLYMORPHISM IN ARENICOLA
MARINA
Structural models have been proposed for the giant extracellular
hemoglobin of A. marina based on the complementarities
of ESI-MS and MALLS [83], on ESI-MS under
non-denaturing conditions [71] and from crystallographic
data [81], all established from a pool of lugworm blood
(Fig. 1). They concluded to different structural models;
AmHb dodecamers subassemblies is either M 3 T 3 or M 9 T,
with or without a central unit [83]. Nowadays, no such accurate
method as ESI-MS exists to validate one structural
model. However, they might be all correct. Indeed, very recent
results obtained on AmHb analyzed individually by
ESI-MS, have revealed the existence of a structural polymorphism
[92].
The extracellular Hb are invariably synthesized intracellularly
and are then secreted. Because they lack the cellular
microenvironment where effector levels maybe regulated as
invertebrates, their operating conditions are more dependent
on the vagaries of ambient conditions than the Hbs enclosed
in ion regulating cells. Invertebrates abundantly display Hbs
heterogeneity that involves both multiplicity (different
isoHbs occurring in the same individual organisms) and
polymorphism (different Hbs components, Hbs patterns, or
relative concentrations occurring in different genetic strains).
This phenomenon is well known for branchiopod groups
[128] but has never been evidenced in annelids so far. The
preliminary observations for AmHb suggest that it is not a
differential gene expression but rather a genetic polymorphism
[92]. This structural polymorphism was evidenced
thanks to the ESI-MS mass accuracy which allowed to evidence
different globin chains and subunits composition
among AmHb taken individually while the individuals were
collected under exactly the same conditions.
Fig. (10) shows 3 different and characteristic MaxEnt
processed spectra of unreduced and reduced AmHb obtained
from 20 A. marina from the same population. Table 4 summarizes
the three different profiles and highlights the possible
existence of a genetic polymorphism with homozygoteand
heterozygote-like profiles as detailed in Fig. (10) and
Table 4. This assumption is actually under investigation
however preliminary mRNA results seems to confirm this
fact [92]. The resolution of ESI-MS under non-denaturing
conditions and of the SEC-MALLS does not allow to evidence
the existence of a structural polymorphism from the
mass of the dodecamers subunits or from the entire HBL-
Hbs.
The results obtained here revealed that the structure of
the AmHb is not unique but different possible models exist
for the species. However, HBL-Hb are always composed of
the association of globin monomer and globin disulfidebound
trimers. Today, we can also imagine that blood of one
A. marina individual is composed of several structurally different
HBL-Hb.
9. INVESTIGATING THE ROLE OF PHENOTYPIC
PLASTICITY IN RESPONSE TO ENVIRONMENTAL
CHALLENGE AT THE MACROMOLECULAR AND
SUBUNIT LEVELS
9.1. Monitoring Aggregation State of Crustacean Hc
As crustacean Hc can exist under several aggregation
states in some groups, the knowledge of their proportions
and how these evolve between different environmental conditions
or between different species is of interest for physiological
studies [106, 129]. The aggregation states of the respiratory
pigment from an individual can be rapidly determined
by SEC-MALLS. A spectrophotometric monitoring at
340 nm enables to discriminate between the pigment and
other non-respiratory proteins.
A physiological buffer is used for elution to maintain the
same chemical environment as in the animal as far as we can
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