Ice-laden Seawater Research Articles

Isolation and characterization of type I antifreeze proteins from cunner,Tautogolabrus adspersus, order Perciformes

Antifreeze proteins (AFPs) are produced by many species of teleost fish that inhabit potentially lethal ice-laden seawater and afford them protection from freezing. To date type I AFPs have been fully characterized in two teleost orders: Pleuronectiformes and Scorpaeniformes. In this study, we report the isolation and complete characterization of a type I AFP present in fish from a third order: cunner (Tautogolabrus adspersus), order Perciformes (family Labridae). This protein was purified from blood plasma and found to belong to what is now known as classical type I AFP with their small size (mass 4095.16 Da), alanine richness (> 57 mol%), high α-helicity (> 99%) with the ability to undergo reversible thermal denaturation, 11 amino acid (ThrX(10)) repeat regions within the primary structure, the capacity to impart a hexagonal bipyramidal shaping to ice crystals and the conservation of an ice-binding site found in many of the other type I AFPs. Partial de novo sequencing of the plasma AFP accounted for approximately half of the peptide mass. Sequencing of a combined liver and skin cDNA library indicated that the protein is produced without a signal sequence. In addition the translated product of the AFP cDNA suggests that it codes for the AFP isolated from plasma. These results further solidify the hypothesis that type I AFPs are multiphyletic in origin and suggest that they represent remarkable examples of convergent evolution within three orders of teleost fish.

Biochemical Adaptation to the Freezing Environment - the Biology of Fish Antifreeze Proteins

Many organisms are known to survive in icy environments. These include both over wintering terrestrial insects and plants as well the marine fish inhabiting high latitudes. The adaptation of these organisms is both a fascinating and important topic in biology. Marine teleosts in particular, can encounter ice-laden seawater that is approximately <TEX>$1^{\circ}C$</TEX> colder than the colligative freezing point of their body fluids. These animals produce a unique group of proteins, the antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) that absorb the ice nuclei and prevent ice crystal growth. Presently, there are at least four different AFP types and one AFGP type that are isolated from a wide variety of fish. Despite their functional similarity, there is no apparent common protein homology or ice-binding motifs among these proteins, except that the surface-surface complementarity between the protein and ice are important for binding. The remarkable diversity of these proteins and their odd phylogenetic distribution would suggest that these proteins might have evolved recently in response to sea level glaciations just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Winter flounder, Pleuronectes americanus, has been used as a popular model to study the regulation of AFP gene expression. It has a built-in annual cycle of AFP expression controlled negatively by the growth hormone. The signal transduction pathways, transcription factors and promoter elements involved in this process have been studied in our laboratory and these studies will be presented.

Antifreeze proteins of teleost fishes.

Marine teleosts at high latitudes can encounter ice-laden seawater that is approximately 1 degrees C colder than the colligative freezing point of their body fluids. They avoid freezing by producing small antifreeze proteins (AFPs) that adsorb to ice and halt its growth, thereby producing an additional non-colligative lowering of the freezing point. AFPs are typically secreted by the liver into the blood. Recently, however, it has become clear that AFP isoforms are produced in the epidermis (skin, scales, fin, and gills) and may serve as a first line of defense against ice propagation into the fish. The basis for the adsorption of AFPs to ice is something of a mystery and is complicated by the extreme structural diversity of the five antifreeze types. Despite the recent acquisition of several AFP three-dimensional structures and the definition of their ice-binding sites by mutagenesis, no common ice-binding motif or even theme is apparent except that surface-surface complementarity is important for binding. The remarkable diversity of antifreeze types and their seemingly haphazard phylogenetic distribution suggest that these proteins might have evolved recently in response to sea level glaciation occurring just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Not surprisingly, the expression of AFP genes from different origins can also be quite dissimilar. The most intensively studied system is that of the winter flounder, which has a built-in annual cycle of antifreeze expression controlled by growth hormone (GH) release from the pituitary in tune with seasonal cues. The signal transduction pathway, transcription factors, and promoter elements involved in this process are just beginning to be characterized.

NMR structural studies on antifreeze proteins

Antifreeze proteins (AFPs) are a structurally diverse class of proteins that bind to ice and inhibit its growth in a noncolligative manner. This adsorption-inhibition mechanism operating at the ice surface results in a lowering of the (nonequilibrium) freezing point below the melting point. A lowering of ~1°C, which is sufficient to prevent fish from freezing in ice-laden seawater, requires millimolar AFP levels in the blood. The solubility of AFPs at these millimolar concentrations and the small size of the AFPs (typically 3-15 kDa) make them ideal subjects for NMR analysis. Although fish AFPs are naturally abundant, seasonal expression, restricted access to polar fishes, and difficulties in separating numerous similar isoforms have made protein expression the method of choice for producing AFPs for structural studies. Expression of recombinant AFPs has also facilitated NMR analysis by permitting isotopic labeling with 15N and 13C and has permitted mutations to be made to help with the interpretation of NMR data. NMR analysis has recently solved two AFP structures and provided valuable information about the disposition of ice-binding side chains in a third. The potential exists to solve other AFP structures, including the newly described insect AFPs, and to use solid-state NMR techniques to address fundamental questions about the nature of the interaction between AFPs and ice.Key words: NMR spectroscopy, antifreeze, ice-binding affinity, review.

NMR structural studies on antifreeze proteins.

Antifreeze proteins (AFPs) are a structurally diverse class of proteins that bind to ice and inhibit its growth in a noncolligative manner. This adsorption-inhibition mechanism operating at the ice surface results in a lowering of the (nonequilibrium) freezing point below the melting point. A lowering of approximately 1 degree C, which is sufficient to prevent fish from freezing in ice-laden seawater, requires millimolar AFP levels in the blood. The solubility of AFPs at these millimolar concentrations and the small size of the AFPs (typically 3-15 kDa) make them ideal subjects for NMR analysis. Although fish AFPs are naturally abundant, seasonal expression, restricted access to polar fishes, and difficulties in separating numerous similar isoforms have made protein expression the method of choice for producing AFPs for structural studies. Expression of recombinant AFPs has also facilitated NMR analysis by permitting isotopic labeling with 15N and 13C and has permitted mutations to be made to help with the interpretation of NMR data. NMR analysis has recently solved two AFP structures and provided valuable information about the disposition of ice-binding side chains in a third. The potential exists to solve other AFP structures, including the newly described insect AFPs, and to use solid-state NMR techniques to address fundamental questions about the nature of the interaction between AFPs and ice.

Antifreeze proteins in the urine of marine fish

Several species of marine teleosts have evolved blood plasma antifreeze polypeptides which enable them to survive in ice-laden seawater. Four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence and secondary structure are currently known. Although all of these antifreezes are relatively small (2.6-33 kd) it was generally thought that they were excluded from the urine by a variety of glomerular mechanisms. In the present study antifreeze polypeptides were found in the bladder urine of winter flounder (Pseudopleuronectes americanus), sea raven (Hemitripterus americanus), ocean pout (Macrozoarces americanus) and Atlantic cod (Gadus morhua). Since the plasma of each of these fish contains a different antifreeze class it would appear that all four classes of antifreeze can enter the urine. The major antifreeze components in the urine of winter flounder were found to be identical to the major plasma components in terms of high performance liquid chromatography retention times and amino acid composition. It is concluded that plasma antifreeze peptides need not be chemically modified before they can enter the urine.

Fish antifreeze proteins: physiology and evolutionary biology

Many marine teleosts have adapted to ice-laden seawater by evolving antifreeze proteins and glycoproteins. These proteins are synthesized in the liver for export to the blood where they circulate at levels of up to 20 mg/mL. There are at least four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence, and secondary structure. In addition to antifreeze structural diversity, fish species differ considerably with respect to mechanisms controlling seasonal regulation of plasma antifreeze concentrations. Some species synthesize antifreeze proteins immediately before the onset of freezing conditions, some synthesize them in response to such conditions, whereas others possess high concentrations all year. Endogenous rhythms, water temperature, photoperiod, and pituitary hormones have all been implicated as regulators of plasma antifreeze protein levels. The structural diversity of antifreeze proteins and their occurrence in a wide range of fish species suggest that they evolved separately and recently during Cenozoic glaciation. Invariably, the genes coding for these antifreeze proteins are amplified, sometimes as long tandem arrays, suggesting intense selective pressure to produce large amounts of protein. The distribution of antifreeze gene types among fish species suggests that they could serve as important tools for studying phylogenetic relationships.

Wolffish antifreeze protein genes are primarily organized as tandem repeats that each contain two genes in inverted orientation.

The antifreeze protein genes of the wolffish (Anarhichas lupus) constitute a large multigene family of 80 to 85 copies, which can be classified into two sets. One-third of the genes were linked but irregularly spaced. The other two-thirds were organized as 8-kilobase-pair (kbp) tandem direct repeats that each contained two genes in inverted orientation; DNA sequence analysis suggests that both genes are functional. Except for a single region specific to each gene, the genes and their immediate flanking sequences were 99.2% identical. This degree of identity ended soon after a putative transcription termination sequence; as the 3' ends of the genes were only 1.3 kbp apart, these sequences might confer mutual protection from interference by transcriptional runoff. A Southern blot of wolffish DNA restricted with enzymes that do not cut within the tandem repeats indicated that the repeats were clustered in groups of six or more. The organization of antifreeze protein genes in the wolffish was very similar to that in the unrelated winter flounder, which produces a completely different antifreeze. This similarity might reflect common dynamics by which their progenitors adapted to life in ice-laden sea water.

Wolffish antifreeze protein genes are primarily organized as tandem repeats that each contain two genes in inverted orientation.

The antifreeze protein genes of the wolffish (Anarhichas lupus) constitute a large multigene family of 80 to 85 copies, which can be classified into two sets. One-third of the genes were linked but irregularly spaced. The other two-thirds were organized as 8-kilobase-pair (kbp) tandem direct repeats that each contained two genes in inverted orientation; DNA sequence analysis suggests that both genes are functional. Except for a single region specific to each gene, the genes and their immediate flanking sequences were 99.2% identical. This degree of identity ended soon after a putative transcription termination sequence; as the 3' ends of the genes were only 1.3 kbp apart, these sequences might confer mutual protection from interference by transcriptional runoff. A Southern blot of wolffish DNA restricted with enzymes that do not cut within the tandem repeats indicated that the repeats were clustered in groups of six or more. The organization of antifreeze protein genes in the wolffish was very similar to that in the unrelated winter flounder, which produces a completely different antifreeze. This similarity might reflect common dynamics by which their progenitors adapted to life in ice-laden sea water.

Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces americanus.

The ocean pout (Macrozoarces americanus) produces a set of antifreeze proteins that depresses the freezing point of its blood by binding to, and inhibiting the growth of, ice crystals. The amino acid sequences of all the major components of the ocean pout antifreeze proteins, including the immunologically distinct QAE component, have been derived by Edman degradation. In addition, sequences of several minor components were deduced from DNA sequencing of cDNA and genomic clones. Fifty percent of the amino acids are perfectly conserved in all these proteins as well as in two homologous sequences from the distantly related wolffish. Several of the conserved residues are threonines and asparagines, amino acids that have been implicated in ice binding in the structurally unrelated antifreeze protein of the righteye flounders. Aside from minor differences in post-translational modifications, heterogeneity in antifreeze protein components stems from amino acid differences encoded by multiple genes. Based on genomic Southern blots and library cloning statistics there are 150 copies of the 0.7-kilobase-long antifreeze protein gene in the Newfoundland ocean pout, the majority of which are closely linked but irregularly spaced. A more southerly population of ocean pout from New Brunswick in which the circulating antifreeze protein levels are considerably lower has approximately one-quater as many antifreeze protein genes. Thus, there appears to be a correlation between gene dosage and antifreeze protein levels, and hence the ability to survive in ice-laden seawater. Southern blot comparison of the two populations indicates that the differences in gene dosage were not generated by a simple set of deletions/duplications. They are more likely to be the result of differential amplification.

Evidence for Antifreeze Protein Gene Transfer in Atlantic Salmon (Salmo salar)

Atlantic salmon (Salmo salar) freeze to death if they come into contact with ice at water temperatures below −0.7 °C. Consequently, sea-pen culture of this species in cold water is severely limited. Winter flounder (Pseudopleuronectes americanus) survive in ice-laden seawater by producing a set of antifreeze polypeptides (AFP). We are attempting to make the Atlantic salmon more freeze resistant by transferring antifreeze protein genes from the winter flounder to the genome of the salmon. Salmon eggs were microinjected with linearized DNA after fertilization. Individual fingerlings (1–2 g) were analyzed for flounder AFP genes by genomic Southern blotting. DNA from 2 out of 30 fingerlings showed hybridization to the flounder DNA probe. Hybridization bands following cleavage by restriction enzymes Sst l and Bam HI were identical to those of the injected DNA. Hybridization following Hind III digestion indicated that the flounder AFP gene was linked to the salmon genome. These hybridization signals were absent in the DNA from control fish. The intensity of the hybridization signals indicated that there was on average at least one copy of the AFP gene present per cell.

FISH GLYCOPEPTIDE AND PEPTIDE ANTIFREEZES : THEIR INTERACTION WITH ICE AND WATER

Glycopeptide and peptide antifreeze agents are present in the body fluids of polar fishes and allow them to avoid freezing in ice-laden seawater. These antifreezes lower the freezing point 200 times more than predicted by colligative relations, but have little effect on the melting point of ice. They bind to ice and appear to inhibit growth by increasing the curvature of growth steps on the ice crystal surface. Such a growth would result in a substantial increase in the roughness of the surface of the crystal. Laser light scattering techniques have been used to evaluate the dependence on temperature of the surface roughness at the ice/antifreeze solution interface. Results show a dramatic increase in scattered light as the temperature is decreased toward the freezing point of antifreeze solutions.

Antifreeze peptides confer freezing resistance to fish

It has been widely accepted that plasma antifreeze proteins are directly responsible for the ability of many marine teleosts to survive in ice-laden seawater. However, there appears to be no direct experimental evidence to indicate that this assumption is correct. In the present study winter flounder (Pseudopleuronectes americanus) showed seasonal changes in freezing resistance that were quantitatively the same as the seasonal changes in plasma antifreeze protein levels. Moreover, when winter flounder antifreeze proteins were injected into rainbow trout (Salmo gairdneri) (a species that does not normally possess antifreeze proteins) they increased the freezing resistance of the trout in direct proportion to plasma antifreeze protein levels attained. These studies indicate that antifreeze proteins are directly responsible for the ability of many marine teleosts to survive icy seawater at temperatures below the colligative freezing points of their blood. There appears to be no requirement for species-specific antifreeze protein receptors in the fish in order for them to act.

Physiology and ecology of notothenioid fishes of the Ross Sea

Abstract Perciform fishes of the suborder Notothenioidei are the dominant fishes in the Ross Sea and in the Southern Ocean in general. They have evolved physiological adaptations which ensure their survival in ice-laden seawater at a temperature of -1.9°C and extended their habitat range. Among these adaptations are relatively high metabolic rates made possible by enzymes efficient in catalyzing energyproducing reactions at subzero temperatures; a restricted range of temperature tolerance; slow growth rates in spite of relatively high metabolic rates; antifreeze glycoproteins which reduce the freezing point of body fluids below that of seawater and prevent ice crystal growth; aglomerular nephrons which preclude the loss of small molecular weight antifreeze molecules in the urine and mechanisms (especially skeletal reduction and lipid deposition) promoting neutral buoyancy in some species even though swim bladders are absent. Species with different buoyancies are associated with the pelagic, cryopelagic, b...