A re-evaluation of the role of type IV antifreeze protein

A lipoprotein-like antifreeze protein (type IV AFP) has previously been isolated only from the blood plasma of the longhorn sculpin. However, the plasma antifreeze activity in all individuals of this species tested from Newfoundland and New Brunswick waters ranges from low to undetectable. A close relative of the longhorn sculpin, the shorthorn sculpin, does have appreciable antifreeze activity in its blood but this is virtually all accounted for by the α-helical, alanine-rich type I AFP, other isoforms of which are also present in the skin of both fishes. We have characterized a putative ortholog of type IV AFP in shorthorn sculpin by cDNA cloning. This 12.2-kDa Gln-rich protein is 87% identical to the longhorn sculpin’s type IV AFP. Recombinant versions of both orthologs were produced in bacteria and shown to have antifreeze activity. Immunoblotting with antibodies raised to type IV AFP shows this protein present in longhorn sculpin plasma at levels of less than 100 μg/mL, which are far too low to protect the blood from freezing at the temperature of icy seawater. This confirms the results of direct antifreeze assays on the plasmas. It appears that type IV AFP has the potential to develop as a functional antifreeze in these fishes but may not have been selected for this role because of the presence of type I AFP. Consistent with this hypothesis is the observation that the type IV AFP gene has not been amplified the way functional antifreeze protein genes have in all other species examined.     

Isolation and characterization of skin-type, type I antifreeze polypeptides from the longhorn sculpin, Myoxocephalus octodecemspinosus

The antifreeze polypeptides (AFPs) are found in several marine fish and have been grouped into four distinct biochemical classes (type I-IV). Recently, the new subclass of skin-type, type I AFPs that are produced intracellularly as mature polypeptides have been identified in the winter flounder (Pleuronectes americanus) and the shorthorn sculpin (Myoxocephalus scorpius). This study demonstrates the presence of skin-type AFPs in the longhorn sculpin (Myoxocephalus octodecemspinosus), which produces type IV serum AFPs. Using polymerase chain reaction-based methods, a clone that encoded for a type I AFP was identified. The clone lacked a signal sequence, indicating that the mature polypeptide is produced in the cytosol. A recombinant protein was produced in Escherichia coli and antifreeze activity was characterized. Four individual Ala-rich polypeptides with antifreeze activity were isolated from the skin tissue. One polypeptide was completely sequenced by tandem MS. This study provides the first evidence of a fish species that produces two different biochemical classes of antifreeze proteins (type I and type IV), and enforces the notion that skin-type AFPs are a widespread biological phenomenon in fish.     

Solution structure of a recombinant type I sculpin antifreeze protein

We have determined the solution structure of rSS3, a recombinant form of the type I shorthorn sculpin antifreeze protein (AFP), at 278 and 268 K. This AFP contains an unusual sequence of N-terminal residues, together with two of the 11-residue repeats that are characteristic of the type I winter flounder AFP. The solution conformation of the N-terminal region of the sculpin AFP has been assumed to be the critical factor that results in recognition of different ice planes by the sculpin and flounder AFPs. At 278 K, the two repeats units (residues 11-20 and 21-32) in rSS3 form a continuous alpha-helix, with the residues 30-33 in the second repeat somewhat less well defined. Within the N-terminal region, residues 2-6 are well defined and helical and linked to the main helix by a more flexible region comprising residues A7-T11. At 268 K the AFP is overall more helical but retains the apparent hinge region. The helical conformation of the two repeats units is almost identical to the corresponding repeats in the type I winter flounder AFP. We also show that while tetracetylated rSS3 has antifreeze activity comparable to the natural AFP, its overall structure is the same as that of the unacetylated peptide. These data provide some insight into the structural determinants of antifreeze activity and should assist in the development of models that explain the recognition of different ice interfaces by the sculpin and flounder type I AFPs.     

Analysis of shorthorn sculpin antifreeze protein stereospecific binding to (2-1 0) faces of ice.

In this paper we report the results of our studies on the stereospecific binding of shorthorn sculpin antifreeze protein (AFP) to (2 -1 0) secondary prism faces of ice. Using ice crystal growth and etching techniques together with molecular modeling, molecular dynamics, and energy minimization, we explain the nature of preferential binding of shorthorn sculpin AFP along the [1 2 2] direction on (2- 1 0) planes. In agreement with ice etching studies, the mechanism of preferential binding suggested by molecular modeling explains why the binding of shorthorn sculpin AFP occurs along [1 2 2] and not along its mirror symmetry-related direction [-1 -2 2] on (2 -1 0). This binding mechanism is based on the protein-crystal surface enantioselective recognition that utilizes both alpha-helical protein backbone matching to the (2 -1 0) surface topography and matching of side chains of polar/charged residues with specific water molecule positions in the ice surface. The mechanisms of winter flounder and shorthorn sculpin antifreeze binding to ice are compared.     

Structures of shorthorn sculpin antifreeze polypeptides

The amino acid sequences of the two major antifreeze polypeptides (AFP) from the shorthorn sculpin have been determined using an automatic protein sequencer and enzymic digestion. These two polypeptides, SS-3 and SS-8, consist of 33 and 45 amino acid residues respectively. The N-terminal methionyl residue is blocked in both the polypeptides. When aligned for maximum structural similarity these two AFP are 80% homologous, and there appears a deletion of 12 amino acid residues at the N-terminal portion of SS-3. Like the winter flounder AFP, both the sculpin AFP also contain the 11-amino-acid repeat sequences. The secondary structure of the sculpin AFP is mainly alpha-helical as deduced from circular dichroic spectral data. The helical content of SS-8 is high (73%), while that of SS-3 is moderate (about 45%). The latter exhibits a relatively weak antifreeze activity. Removal of the blocked N-terminal residue in SS-8 did not alter the helical content significantly but did reduce the antifreeze activity. Helical contents of proteolytically generated fragments of AFP are much lower, and they are devoid of activity. The alpha-helix in the SS-8 component is seen to be amphiphilic in character. The relevance of this feature to the mechanism of the antifreeze action is briefly discussed.     

Type I shorthorn sculpin antifreeze protein: recombinant synthesis, solution conformation, and ice growth inhibition studies

A number of structurally diverse classes of "antifreeze" proteins that allow fish to survive in sub-zero ice-laden waters have been isolated from the blood plasma of cold water teleosts. However, despite receiving a great deal of attention, the one or more mechanisms through which these proteins act are not fully understood. In this report we have synthesized a type I antifreeze polypeptide (AFP) from the shorthorn sculpin Myoxocephalus scorpius using recombinant methods. Construction of a synthetic gene with optimized codon usage and expression as a glutathione S-transferase fusion protein followed by purification yielded milligram amounts of polypeptide with two extra residues appended to the N terminus. Circular dichroism and NMR experiments, including residual dipolar coupling measurements on a 15N-labeled recombinant polypeptide, show that the polypeptides are alpha-helical with the first four residues being more flexible than the remainder of the sequence. Both the recombinant and synthetic polypeptides modify ice growth, forming facetted crystals just below the freezing point, but display negligible thermal hysteresis. Acetylation of Lys-10, Lys-20, and Lys-21 as well as the N terminus of the recombinant polypeptide gave a derivative that displays both thermal hysteresis (0.4 degrees C at 15 mg/ml) and ice crystal faceting. These results confirm that the N terminus of wild-type polypeptide is functionally important and support our previously proposed mechanism for all type I proteins, in which the hydrophobic face is oriented toward the ice at the ice/water interface.     

Type I Antifreeze Proteins: Possible Origins from Chorion and Keratin Genes in Atlantic Snailfish

Type I antifreeze proteins (AFPs) are alanine-rich α-helical polypeptides found in some species of right-eye flounders, sculpin, and snailfish. In this study, a shorthorn sculpin skin type I cDNA clone was used to probe an Atlantic snailfish liver cDNA library in order to locate expressed genes corresponding to snailfish plasma AFPs. Clones isolated from the cDNA library had sections with substantial amino acid and nucleotide sequence similarity to snailfish type I AFPs. However, further analysis revealed that the positives were actually three different liver-expressed proteins—two were eggshell proteins, while the third was a type II keratin. We propose that a shift in reading frame could produce alanine-rich candidate AFPs with possible antifreeze activity or ice crystal modification properties. Furthermore, it is plausible that one or more of the liver-expressed proteins represent the progenitors of snailfish type I AFPs. [Reviewing Editor: Dr. John Oakeshott]     

On the low viscosity blood of two cold water,marine sculpins

Summary The viscosities of blood from shorthorn sculpin (Myoxocephalus scorpius), longhorn sculpin (Myoxocephalus octodecemspinosus) and winter flounder (Pseudopleuronectes americanus) were compared using a cone-plate viscometer. Both species of sculpin were almost identical with respect to blood and plasma viscosity at the temperatures (0 and 15°C) and shear rates (2.3–90/s) examined. In contrast, the viscosities of winter flounder blood and plasma were considerably greater than those observed in the sculpins. This difference in blood viscosity between the shorthorn sculpin and the winter flounder persisted over the hematocrit range of 0 to 40% red blood cells. The viscosity of the plasma and the interactions between plasma proteins and red blood cells appeared to be the major reasons for the relatively high viscosity of the flounder blood. Although a proportion of the flounder blood viscosity was attributable to fibrinogen, other plasma proteins also appeared to play a significant role. The relatively low blood viscosity of the sculpin species may confer a circulatory advantage during periods of low water temperatures.     

Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components

Summary Eight major antifreeze polypeptides (AFP) were purified from the sera of Newfoundland ocean pout. Except for their approximately identical size (6,000 Dalton), these components were shown to be separate entities by their behaviour on polyacrylamide gel electrophoresis, ion exchange chromatography, gel permeation and reverse phase high performance liquid chromatography. They could also be divided into two cross-reactive, yet distinct, immunological groups. Amino acid analysis demonstrated that ocean pout AFP are different from all of the other antifreezes studied to date. The ocean pout AFP do not contain the abundance of alanine (60 mol%) found in winter flounder and shorthorn sculpin AFP nor the high half-cystine residues (8 mol%) observed in sea raven AFP. It is suggested that ocean pout AFP represent a new type of macromolecular antifreeze.Abbreviations AFGP antifreeze glycoprotein(s) - AFP antifreeze polypeptide(s) - HPLC high performance liquid chromatography - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis     

Molecular and comparative analyses of type IV antifreeze proteins (AFPIVs) from two Antarctic fishes, Pleuragramma antarcticum and Notothenia coriiceps

Antifreeze protein type IV (AFPIV) cDNAs and genomic DNAs from the Antarctic fishes Pleuragramma antarcticum (Pa) and Notothenia coriiceps (Nc) were cloned and sequenced, respectively. Each cDNA encoded 128 amino acids, with 94% similarity between the two and 83% similarity with AFPIV of the longhorn sculpin, Myoxocephalus octodecemspinosus. The genome structures of both genes consisted of four exons and three introns, and were highly conserved in terms of sequences and positions. In contrast, the third intron of PaAFPIV had additional nucleotides with inverted repeats at each end, which appeared to be a MITE-like transposable element. Comparative analysis revealed that fish AFPIVs were widely distributed across teleost fishes, well conserved in their intron positions, but more variable in intron sequences and sizes. However, the intron sequences of two Antarctic fishes were highly conserved, indicating recent radiation of notothenioids in the evolutionary lineage. The recombinant PaAFPIV and NcAFPIV were expressed in E. coli, and examined antifreeze activity. PaAFPIV and NcAFPIV gave ice crystals with star-shaped morphology, and thermal hysteresis (TH) values were 0.08°C at the concentration of 0.5mg/ml.     

Type I antifreeze proteins expressed in snailfish skin are identical to their plasma counterparts

Type I antifreeze proteins (AFPs) are usually small, Ala-rich alpha-helical polypeptides found in right-eyed flounders and certain species of sculpin. These proteins are divided into two distinct subclasses, liver type and skin type, which are encoded by separate gene families. Blood plasma from Atlantic (Liparis atlanticus) and dusky (Liparis gibbus) snailfish contain type I AFPs that are significantly larger than all previously described type I AFPs. In this study, full-length cDNA clones that encode snailfish type I AFPs expressed in skin tissues were generated using a combination of library screening and PCR-based methods. The skin clones, which lack both signal and pro-sequences, produce proteins that are identical to circulating plasma AFPs. Although all fish examined consistently express antifreeze mRNA in skin tissue, there is extreme individual variation in liver expression - an unusual phenomenon that has never been reported previously. Furthermore, genomic Southern blot analysis revealed that snailfish AFPs are products of multigene families that consist of up to 10 gene copies per genome. The 113-residue snailfish AFPs do not contain any obvious amino acid repeats or continuous hydrophobic face which typify the structure of most other type I AFPs. These structural differences might have implications for their ice-crystal binding properties. These results are the first to demonstrate a dual liver/skin role of identical type I AFP expression which may represent an evolutionary intermediate prior to divergence into distinct gene families.     

Activity of short segments of Type I antifreeze protein

In this work, we present a study on the antifreeze activity of short segments of a Type I antifreeze protein, instead of the whole protein. This approach simplifies the correlation between antifreeze protein characteristics, such as hydrophilicity/hydrophobicity, and the effect of these characteristics on the antifreeze mechanism. Three short polypeptides of Type I AFP have been synthesized. Their antifreeze activity and interactions with water and ice crystals have been analyzed by various techniques such as circular dichroism spectroscopy, X-ray diffraction, differential scanning calorimetry, and osmometry. It is shown that one short segment of Type I AFP has an antifreeze activity of about 60% of the native protein activity. In this work, we demonstrate that short segments of Type I AFPs possess nonzero thermal hysteresis and result in modifications in the growth habits and growth rates of ice. This approach enables the preparation of large quantities of short AFP segments at low cost with high antifreeze activity, and opens the possibility of developing the commercial potential of AFPs.     

High concentrations of methemoglobin in five species of temperate marine teleosts

Blood samples from five species of marine teleosts were assayed for methemoglobin (metHb) levels during winter and summer acclimatization. There was at least 7% total hemoglobin in the met-form in all species, and as high as 27% in one species, the Atlantic cod (Gadus morhua). There was significant seasonal variation in metHb levels for three of the five species, the highest values occurring during the winter months; cunners (Tautogolabrus adspersus) 15.6% in winter and 10.1% in the summer, shorthorn sculpin (Myoxocephalus scorpius) 20.0% in the winter and 8.19% in the summer, longhorn sculpin (Myoxocephalus octodecemspinosus) 17.3-21.6% in the winter and 8.12% in the summer. The winter flounder (Pseudopleuronectes americanus) and the Atlantic cod maintained metHb concentrations constant throughout the year: 13% and 27%, respectively. There does not appear to be any relationship between the activity of a fish and the level of metHb in its blood.     

Hypothermic protection--a fundamental property of "antifreeze" proteins

For the last two decades fish antifreeze proteins have been considered to function exclusively in conferring freeze-resistance to fish by binding to ice crystals and thereby depressing blood plasma freezing points non-colligatively. We report here the discovery of a second fundamental property of antifreeze proteins, the ability to protect cells and their membranes from hypothermic damage. Experiments were carried out exposing immature bovine oocytes to 4 degrees C for 24 h in the presence of type I alanine rich alpha helical antifreeze polypeptides (AFP) from winter flounder, type II cysteine-rich AFP from sea raven or type III AFP from ocean pout. The presence of AFP in the incubation medium resulted in an approximate four fold increase in the number of oocytes retaining an intact oolemma and a three fold increase in the number of oocytes able to undergo in vitro maturation. None of the control oocytes could be fertilized, whereas, of those incubated in AFP, the percentage which developed normally following fertilization was comparable to that observed for fresh oocytes. These results indicate that cold-sensitive mammalian cells can be rendered cold-tolerant through the addition of "antifreeze" proteins.     

Helical Antifreeze Proteins Have Independently Evolved in Fishes on Four Occasions

Alanine-rich α-helical (type I) antifreeze proteins (AFPs) are produced by a variety of fish species from three different orders to protect against freezing in icy seawater. Interspersed amongst and within these orders are fishes making AFPs that are completely different in both sequence and structure. The origin of this variety of types I, II, III and antifreeze glycoproteins (AFGPs) has been attributed to adaptation following sea-level glaciations that occurred after the divergence of most of the extant families of fish. The presence of similar types of AFPs in distantly related fishes has been ascribed to lateral gene transfer in the case of the structurally complex globular type II lectin-like AFPs and to convergent evolution for the AFGPs, which consist of a well-conserved tripeptide repeat. In this paper, we examine the genesis of the type I AFPs, which are intermediate in complexity. These predominantly α-helical peptides share many features, such as putative capping structures, Ala-richness and amphipathic character. We have added to the type I repertoire by cloning additional sequences from sculpin and have found that the similarities between the type I AFPs of the four distinct groups of fishes are not borne out at the nucleotide level. Both the non-coding sequences and the codon usage patterns are strikingly different. We propose that these AFPs arose via convergence from different progenitor helices with a weak affinity for ice and that their similarity is dictated by the propensity of specific amino acids to form helices and to align water on one side of the helix into an ice-like pattern.     

Isolation and Identification of Antifreeze Protein with High Activity in Ammopiptanthus mongolicus

The authors have isolated and partial purified antifreeze protein antifreeze protein (AFP) produced endogenously in Arnrnopiptanthus rnongolicus. The results show that the partial purified AFP ranged in size including 45.7 kD, 81.2 kD, and so on. At 50 mg/mL protein concentration, the temperature of melting point is –15 ℃, and its freezing point is even lower. Therefore, the AFP activity exhibited by the Arnmopiptanthus mongolicus is higher than that observed for AFP found in polar fishes or in winter rye. In addition, by a phase contrast light photomicroscope, the author have observed the morphology of individual ice crystals formed in the solution, including squares, rectangles, cones, hexagons. The morphology of these ice crystals are similar to those of ice crystals observed in polar fishes and in cold-acclimation winter rye.     

Epithelial dominant expression of antifreeze proteins in cunner suggests recent entry into a high freeze-risk ecozone

Most marine teleost fishes residing in a high freeze-risk ecozone, such as the coastal waters of Newfoundland during winter, avoid freezing by secreting high concentrations of antifreeze proteins (AFP) into their blood plasma where they can bind to and prevent the growth of ice that enter the fish. Cunner (Tautogolabrus adspersus), which overwinter in such shallow waters are the only known exception. Although this species does produce type I AFP, the plasma levels are too low to be of value as a freeze protectant. Southern and Northern blot analyses carried out in this study establish that the cunner AFP genes belong to a multigene family that is predominantly expressed in external epithelia (skin and gill filaments). These results support the hypothesis that the survival of cunner in icy waters is attributable in part to epithelial AFP that help block ice propagation into their interior milieu. In contrast to the cunner, heterospecifics occupying the same habitat have greater freeze protection because they produce AFP in the liver for export to the plasma as well as in external epithelia. Since the external epithelia would be the first tissue to come into contact with ice it is possible that one of the earliest steps involved in the evolution of freeze resistant fish could have been the expression of AFP in tissues such as the skin. We suggest that this epithelial-dominant AFP expression represents a primitive stage in AFP evolution and propose that cunner began to inhabit “freeze-risk ecozones” more recently than heterospecifics.     

Antifreeze proteins differentially affect model membranes during freezing

Over the past decade antifreeze proteins from polar fish have been shown either to stabilize or disrupt membrane structure during low temperature and freezing stress. However, there has been no systematic study on how membrane composition affects the interaction of antifreeze proteins with membranes under stress conditions. Therefore, it is not possible at present to predict which antifreeze proteins will protect, and which will damage a particular membrane during chilling or freezing. Here, we analyze the effects of freezing on spinach thylakoid membranes and on model membranes of varying lipid composition in the presence of antifreeze protein type I (AFP I) and specific fractions of antifreeze glycoproteins (AFGP). We find that the addition of galactolipids to phospholipid model membranes changes the effect each protein has on the membrane during freezing. However, the greatest differences observed in this study are between the different types of antifreeze proteins. We find that AFP type I and the largest molecular weight fractions of AFGP induce concentration dependent leakage from, and are fusogenic to the liposomes. This is the first report that an antifreeze protein induces membrane fusion. In contrast, the smallest fraction of AFGP offers a limited degree of protection during freezing and does not induce membrane fusion at concentrations up to 10 mg/ml.     

Expression of a cystine-rich fish antifreeze in transgenicDrosophila melanogaster

We have usedDrosophila melanogaster as a model system for the transgenic expression of cystine-rich Type II antifreeze protein (AFP) from sea raven. This protein was synthesized and secreted into fly haemolymph where it migrated as a larger species (16 kDa) than the mature form of the protein (14 kDa) as judged by immunoblotting.Drosophila-produced Type II AFP demonstrated antifreeze activity both in terms of thermal hysteresis (0.13 °C) and inhibition of ice recrystallization. Recombinant AFP was purified and N-terminal sequencing revealed a 17 aa extension that began at the predicted signal peptide cleavage point. The expression of all three AFP types in transgenicDrosophila has now been achieved. We conclude that the globular Type II and Type III AFPs are better choices for antifreeze transfer to other organisms than is the more widely used linear Type I AFP.