Role of Antifreeze Proteins in Freezing Avoidance in Antarctic Fishes: Ecological and Organismal Physiology, Structure-Function and Mechanism, Genetics and Evolution

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9615023 DeVries The project includes on-going and new studies on the role of antifreeze glycopeptides (AFGPs) and peptides (AFPs) in freezing avoidance of Antarctic fishes in five specific areas: (1) relationship of severity of environment and association of ice in fish; (2) the uptake of endogenous ice and its fate; (3) structure-function of antifreeze proteins including the molecular mechanism of AP adsorption and inhibition of ice growth; (4) structures and organizations of AP genes and gene families and their relationship to protein characteristics and gene evolution; (5) tissue specificity of AFGP expression. The extent of exogenous and endogenous ice will be determined for McMurdo area fishes, which experience the coldest and most ice-laden waters of the Antarctic region. The severity of the environment will be investigated by deriving freezing isotherms from CTD data in McMurdo Sound and waters beneath the McMurdo Ice Shelf and correlated with physical and biological measurements of the presence of the ice. Similar experiments will be conducted for the less severe marine environment of the Antarctic Peninsular. These studies will correlate freezing extremes with circulating levels of AFGPs in the fishes associated with these environments. The uptake of endogenous ice and its fate will be determined by experimentally introducing labeled ice into the fish to find out its route of disappearance from the blood and ultimately its fate. Experiments will be designed to test whether macrophages can increase solute concentration and "melt" or dissolve ice crystals. The relationship of AP structure to function will be determined for some of the eel pout AFPs. Detailed structural information will be obtained from X-ray crystallography and computer aided modeling to determine the nature of the lattice match between the surface polar residues and the water molecules in the ice lattice on the adsorption plane. A new unique AP will be characterized in the Antarctic smelt Pleuragramma antarcticum and its ice crystal size-dependent antifreeze activity investigated. Molecular characterizations of AFGP and APP genes and gene families, and their arrangement in the genome will be carried out to further our understanding of the genetic parameters governing gene dosage versus environmental exigency, protein abundance, complex protein heterogeneity, and the evolution of these gene families. Cellular and molecular aspects of the expression of AFGPs in pancreas, the newly discovered major synthetic tissue, will be investigated.