Characterization of skin and plasma type I antifreeze proteins from Atlantic (Liparis atlanticus) and dusky (Liparis gibbus) snailfish

Evans, Robert Philip (2003) Characterization of skin and plasma type I antifreeze proteins from Atlantic (Liparis atlanticus) and dusky (Liparis gibbus) snailfish. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Atlantic snailfish (Liparis atlanticus) and dusky snailfish (Liparis gibbus) belong to a large family of benthic and pelagic marine fishes that inhabit northern regions of the Atlantic Ocean. Both species spawn during the winter months in ice-laden inshore coastal regions around Newfoundland. Due to their harsh winter environment, snailfish are prime candidates for production of antifreeze proteins (AFPs). -- Initial results confirmed that plasma from both species contain alanine rich, α-helical, type I AFPs that are significantly larger (>9.3 kDa) than all previously described type I AFPs. Surprisingly, their skin tissues produce AFPs that are identical to those which circulate in blood. While all snailfish consistently express antifreeze mRNA in skin tissue, there is extreme individual variation in liver expression - an unusual phenomenon that has never been reported previously. Molecular analyses revealed that snailfish AFPs are products of multi gene families that consist of at least ten gene copies per genome. It is unclear if liver and skin antifreeze mRNAs are expressed by the same gene or a separate subset of genes which is typical of other fish that produce skin-type AFPs. -- Although the 113 residue snailfish AFPs are unusually long, their amino acid composition, highly a-helical secondary structure and the bipyramidal ice-crystals they create are characteristic of all type I AFPs. However, unlike other type I AFPs, snailfish proteins do not contain any obvious amino acid repeats or a continuous hydrophobic face that typify the structure of most other type I AFPs. These structural differences might have implications for their ice crystal binding properties. Biochemical experiments demonstrated that physiological concentrations of normal salts are responsible for a significant increase in thermal hysteresis activity in antifreeze proteins and glycoproteins. The colligative effects of these salts can account for the supplementary freezing point depression of blood required to ensure survival of marine fish in ice-laden seawater during winter. -- Two eDNA clones were identified from a snailfish liver eDNA library that code for fish eggshell proteins while a third one codes for a type II keratin. However, all three of these clones contain sections with substantial amino acid and nucleotide sequence similarity to snailfish type I AFPs. It is plausible that one or more of these proteins represent the ancestral proteins of snailfish type I AFPs. -- Novel type I AFPs were isolated and partially characterized from skin tissues of cunner (Tautogolabrus adspersus). Type II AFPs that are identical to those expressed in liver for export into blood were isolated from sea raven (Hemitripterus americanus) skin tissue extracts. Taken together the data generated in this thesis has strengthened and widened the scope of the hypothesis that skin represents the primordial source of AFP expression. It is clear that epithelial tissues are a primarily important source for antifreeze expression to enhance the complement of AFPs that protect fish from freezing in extreme cold environments.

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