Functional and comparative studies of haemoproteins from polar fishes
Riccio, Alessia (2011) Functional and comparative studies of haemoproteins from polar fishes. [Tesi di dottorato] (Inedito)
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Proteins, such as the members of the globin superfamily, are sensitive to temperature and their properties are the result of a long process of adaptation to the conditions encountered during the species evolution. The globin superfamily comprises globular proteins that reversibly bind gaseous ligands such as O2, CO and NO to a haem prosthetic group, Fe-protoporphyrin IX. The globins of this family are the components of classical haemoglobin (Hb) and myoglobin (Mb), but also of neuroglobin, cytoglobin (Cygb), globin X, globin Y and eye-globin. In this study, particular attention has been given to biochemical and physico-chemical characterisation of two proteins. On one hand, the O2 transport systems from two Arctic fish species (Lycodes reticulatus and the cod Gadus morhua) have been investigated. On the other, Cygb has been studied from two Antarctic fish species, one belonging to the family Channichthyidae (icefish) lacking Hb and Mb (Chaenocephalus aceratus) and one red-blooded species belonging to the family Nototheniidae (Dissosticus mawsoni). The Arctic and Antarctic regions have the low temperature in common but differ in geographic position and history. The Antarctic is a continent isolated by the Polar Front, a circular oceanic system, and the temperatures are constantly close to -1.87°C. In contrast, the Arctic is essentially an ocean that lies between North America, Greenland, Europe and Asia. There are strong currents with high temperature variations. The Arctic and Antarctic icthyofaunas are very different. In the Antarctic, a single group of teleost fishes is dominant, the suborder Notothenioidei, that includes eight families. The modern family Channichtyidae is particularly interesting because its species have coulorless blood, lacking Hb and in some cases Mb. In contrast, in the Arctic there are six marin groups, nobody being dominant. Given a shorter evolutionary time at polar temperatures, than the Antarctic ichthyofauna, Arctic fish may provide valuable information on the effects of environmental temperature on specific physiological and biochemical traits. It is note that fish Hbs offer the possibility to investigate functional differentiation and molecular adaptations in species living in a large variety of environmental conditions. In this study, the structural and functional characterisation of the hemolysate of L. reticulatus (family Zoarcidae), living on the sea floor near the coasts of northern Europe and North America is reported. The hemolysate shows only a single α chain, whereas polymorphism of two β chains, which differ by only four residues corresponding to two Hbs. For such a high identity, complete purification of the two Hbs was not achieved and the functional studies were carried out on the hemolysate. The latter showed a low Bohr effect and no Root effect. The Hbs tend to form high-molecular mass polymers at physiological pH and low temperature (4°C), as shown by gel-filtration chromatography and dynamic light scattering. The elucidation of the primary structure has allowed to establish correlation between functional behaviour (no Root effect) and structural properties (polymerisation). In fact, it was demonstrated that Cys residues are present in high number and tend to form intermolecular disulphide bridges as shown by mass spectrometry. Recently, an unusual process of Hb polymerisation (sickling), which occurs in vivo in red blood cells of several Arctic species of the family Gadidae, was discovered and reported in the literature. The G. morhua Hb polymerisation showed pH- and concentration-dependence in the deoxygenate state in vitro, suggesting that polymerisation may be an adaptive response to extreme and stressful environmental conditions. Therefore, Arctic fish Hbs appear to be very useful models for studying sickling disorders and Hb-polymerisation processes. The second topic of the thesis were two Cygbs from Antarctic fish. Cygb is a cytoplasmatic protein found in almost all tissues and characterized by endogenous hexacoordination of the haem. The function is not clear. Involvement in protection from oxidative stress, in NO metabolism, in collagen synthesis and in defence mechanisms of cancer cells was hypothesised. Cygb was found in both: in red-blood D. mawsoni and in the icefish C. aceratus. The Cygbs were cloned, expressed and purified and a preliminary characterisation was carried out. It was demonstrated that they are hexacoordinated independently of pH- and temperature, similar to human Cygb. Understanding the role of the Cygb genes in species lacking Hb and Mb is a very important task necessary to elucidate of the function of this protein.
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