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The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein and Peptide Science, 2008, Vol. 9, No. 2 173 groups present a high diversity of polypeptide chains and subassemblies. They have from 2 to 4 monomeric globin chains, except for Eudistylia vancouverii (no monomeric chain) and Tylorrhynchus heterochaetus (one monomeric chain). Concerning linker chains, 3 or 4 different monomeric chains are usually found, except for Paralvinella palmiformis which surprisingly has only one linker chain, thus evidencing that one linker type is sufficient to build HBL-Hb structure [87]. A few polychaete species have linker dimers (Methanoaricia dendrobranchiata, Tylorrhynchus heterochaetus, Arenicola marina). The case of Eudistylia Chl is original since globin chains are arranged in divalent dimers and tetramer and 10 monomeric linker chains are present. Few data are available for other Chl; it would be of interest to analyze other Chl with the same precision in order to test whether such high variability is specific to sabellids or if Eudistylia is an isolated case [70]. As previously reported, groups can be separated on the basis of their globin covalent assemblies [37]. In hirudinids (achaetes), globin chains are monomeric and dimeric and globin dodecamers are made of 6 monomers and 3 dimers. In oligochaetes, globin chains are monomeric and trimeric (except for Glossoscolex paulistus with a small amount of dimer) and dodecamers are made of 3 monomers and 3 trimers. In polychaetes, globin chains are mostly monomeric and trimeric except for Methanoaricia dendrobranchiata, Riftia pachyptila and Tevnia jerichonana which present dimeric chains and sabellids with dimeric and tetrameric globin chains. Interestingly, dimeric globin chains are also observed in the 400 kDa vascular Hb from Riftia pachyptila and the shallow water pogonophoran Oligobrachia mashikoi [68, 86]. These observations suggest that the occurrence of a dimeric globin chain cannot be linked simply to a particular annelid group (present in Palpata and Scolecida) nor to an environmental condition (present in species living in shallow water and deep-sea communities). Thus subassembly structure does not seem to follow simple taxonomic distribution. However general trends can be drawn: hirudinids, oligochaetes and polychaetes have mainly a 6M+3D, 3M+3T and 3M+3T structure, respectively. A number of exceptions exist concerning polychaetes. It is tempting to relate the high diversity within the polychaete group either with its broader taxonomic diversity or with the high diversity of habitats these species live in. Whereas oligochaetes and achaetes are limited to fresh water and terrestrial habitats, polychaetes are found in various and contrasted marine habitats, e.g. intertidal mud, shallow water, deep-sea hydrothermal vents and cold seeps. In addition, annelid phylogeny is still under debate and could be reconsidered in the forthcoming years [112-115]. The high diversity evidenced by ESI-MS analysis also exists at the spatial level, as illustrated by the occurrence of architectural type I or II and toroid or ellipsoid central piece in the different groups (Table 1). However no relation between spatial arrangement and subunit composition can be observed with the available data. ESI-MS has helped to determine precise distribution of subunit among species and to characterize some of their structural features. The global scheme which is depicted is a globally well-conserved organization of HBL-Hb with subunit assemblies that are typical of some groups, whereas some particular features such as dimeric globin chain presence, architectural type and central piece nature seem to follow no phylogenic determination. Fig. (9) presents the comparison between MALLS masses and model masses calculated from a typical LtHb model (144 globins + 36 linkers), from denaturing ESI-MS mass data. MALLS mass is almost always superior to model mass. A slight difference seems consistent with Carcinus maenas data. In C. maenas case, the comparison occurs between MALLS mass and native ESI-MS mass (personal data). The resulting ~2-4 % difference is thus occurring between two molecular species which are certainly the same (native Hc). Three main groups can be observed in Fig. (9b). The first one exhibit mass differences ~2-4 %, which is consistent with C. maenas observed differences and suggests a good agreement between MALLS mass and model. The second group presents mass differences of about 6 to 7 % and the third one is well separated with differences over 10 % (Paralvinella grasslei, Alvinella pompejana, Macrobdella decora and one measurement for Lumbricus terrestris). Several elements can be proposed to explain these differences. As already mentioned, differences below 5% can be thought of as standard (observed for C. maenas for the same structure in MALLS and ESI-MS). Higher differences can mean that the proposed structural model 144 globins + 36 linkers is not well adapted for these species [83, 87, 88]. Daniel and collaborators [116] have proposed that the discrepancy observed in LtHb native masses could be due to the existence of two forms, one form of 4.4 MDa with 192 globin and 36 linker chains, and the classical form of 3.6 MDa with 144 globin and 36 linker chains. They proposed that the 4.4 MDa form would be the truly native form present in the hemolymph corresponding to the 144 + 36 model associated with 48 globins in the central part. The 3.6 MDa form would result from the dissociation of the fragile former one. They support this hypothesis with the observation of Riftia pachyptila V2 hemoglobin of ~430 kDa, composed of 24 globin chains [68, 85]. The observed differences in Fig. (9) could result from a partial degradation of a hypothetical 192 + 36 form in the sample: species with differences 5 % would present a mixture of complete and dissociated HBL-Hbs (192 + 36 and 144 + 36) and thus an average mass would be measured. In this case, the two groups (differences of about 6-7 % and >10 %) could differ in the fragility of their HBL-Hbs. A higher thermostability and resistance to reduction has already been observed for Alvinella pompejana Hb [88, 117]. 7.2. Hc Diversity in Crustaceans Two main features can be studied when investigating Hc diversity in crustaceans: the dodecamer and hexamer proportions and the subunit heterogeneity. These domains have been thoroughly investigated in past decades [106, 118] but to our knowledge MALLS and MS have been applied only a very few times and very recently to this kind of study, even if light scattering was frequently used for investigation of molluscan Hc and sporadically crustacean Hc [119-122]. The determination of aggregation forms in crustacean hemolymph was performed most of the time by using sedi- 94
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 95
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THESE DE DOCTORAT DE L’UNIVERSITE
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i Résumé / Abstract Réponse adap
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iv SOMMAIRE 4.2 Objectifs de l’é
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2 CHAPITRE 1. INTRODUCTION Les éco
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4 CHAPITRE 1. INTRODUCTION 1.1 Mili
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6 CHAPITRE 1. INTRODUCTION Les prin
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8 CHAPITRE 1. INTRODUCTION TAB. 1.1
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10 CHAPITRE 1. INTRODUCTION et cycl
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FIG. 1.5 - Localisation des princip
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(a) FIG. 1.6 - Fonctionnement d’u
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16 CHAPITRE 1. INTRODUCTION TAB. 1.
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18 CHAPITRE 1. INTRODUCTION FIG. 1.
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26 CHAPITRE 1. INTRODUCTION Capacit
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28 CHAPITRE 1. INTRODUCTION TAB. 1.
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30 CHAPITRE 1. INTRODUCTION 700 600
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32 CHAPITRE 1. INTRODUCTION férent
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34 CHAPITRE 1. INTRODUCTION (a) (b)
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144 CHAPITRE 4. PLASTICITÉ PHÉNOT
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Dénaturant Natif
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172 CHAPITRE 5. ADAPTATIONS RESPIRA
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216 CHAPITRE 6. CONCLUSION ET PERSP
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Annexe A Détermination des masses
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229 10000 h280 .M h) h280−h337 80
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Annexe B Détermination des distrib
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233 Modèle 2 : 2 sous-unités lég
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235 1 0.9 0.8 6 s-u aléatoires 2 s
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Bibliographie Adair, G. S. The hemo
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BIBLIOGRAPHIE 239 Catalina, M. I.,
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BIBLIOGRAPHIE 241 Farmer, C. S., J.
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BIBLIOGRAPHIE 243 Hourdez, S. Adapt
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BIBLIOGRAPHIE 245 Mangum, C. P. Sub
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BIBLIOGRAPHIE 247 Rainer, J., C. P.
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BIBLIOGRAPHIE 249 Svedberg, T. et I
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BIBLIOGRAPHIE 251 Vanin, S., E. Neg
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Liste des figures 1.1 Cycle des mar
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LISTE DES FIGURES 255 4.17 Profils
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Table des matières Résumé Sommai
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TABLE DES MATIÈRES 261 4.3.5 Spect
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TABLE DES MATIÈRES 263 Table des m
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