Functional Diversification and Evolution of Antifreeze Proteins in the Antarctic Fish Lycodichthys dearborni

Antifreeze proteins (AFPs) have independently evolved in many organisms. AFPs act by binding to ice crystals, effectively lowering the freezing point. AFPs are often at high copy number in a genome and diversity exists between copies. Type III antifreeze proteins are found in Arctic and Antarctic eel pouts, and have previously been shown to evolve under positive selection. Here we combine molecular and proteomic techniques to understand the molecular evolution and diversity of Type III antifreeze proteins in a single individual Antarctic fish Lycodichthys dearborni. Our expressed sequence tag (EST) screen reveals that at least seven different AFP variants are transcribed, which are ultimately translated into five different protein isoforms. The isoforms have identical 66 base pair signal sequences and different numbers of subsequent ice-binding domains followed by a stop codon. Isoforms with one ice-binding unit (monomer), two units (dimer), and multiple units (multimer) were present in the EST library. We identify a previously uncharacterized protein dimer, providing further evidence that there is diversity between Type III AFP isoforms, perhaps driven by positive selection for greater thermal hysteresis. Proteomic analysis confirms that several of these isoforms are translated and present in the liver. Our molecular evolution study shows that paralogs have diverged under positive selection. We hypothesize that antifreeze protein diversity is an important contributor to depressing the serum freezing point.     

Type II antifreeze protein from a mid-latitude freshwater fish,Japanese smelt (Hypomesus nipponensis)

A lot of reports of antifreeze protein (AFP) from fish have been published, but no report has mentioned of commercialized mid-latitude fresh water fish which producing AFP in its body fluid. We found that the AFP in the body fluid of Japanese smelt (Hypomesus nipponensis) from mid-latitude fresh water was purified and characterized. The N-terminal amino acid sequence of the Japanese smelt AFP was 75.0% identical to Type II AFP from herring. Results of EDTA treatment and ruthenium red staining suggested that the Japanese smelt AFP had at least one Ca2+-binding domain. Interestingly, the antifreeze activity of the Japanese smelt AFP did not completely disappear when Ca2+ ions were removed. The molecular mass of the Japanese smelt AFP was calculated to be 16,756.8 by the TOF-mass analysis. The Open reading flame of the gene coding for the Japanese smelt AFP was 444 bp long and was 85.0% identical with the entire herring AFP gene. The cDNA and amino acid sequence of the Japanese smelt AFP were the same length as those of herring AFP.     

Accumulation of type I fish antifreeze protein in transgenic tobacco is cold-specific

Expression of fish antifreeze protein (AFP) genes in plants is a possible means of increasing their frost resistance and freeze tolerance. Initial work involved transfer into tobacco of an AFP gene from winter flounder which codes for the alanine-rich, -helical Type I AFP. Plants were transformed with a gene construct in which the preproAFP cDNA was inserted between the cauliflower mosaic virus 19S RNA promoter and the nopaline synthetase polyadenylation site. Although transgenic plants produced AFP mRNA, no AFP was detected on western blots. Re-evaluation of AFP expression in these transgenic plants showed that AFP accumulated to detectable levels only after exposure of the plant to cold. Extracts of plants incubated at 4°C for 24 h contained a protein which co-migrated with winter flounder proAFP and was cross-reactive to Type I AFP antisera. Two other minor protein bands of slightly higher apparent M _r also cross-reacted with the antisera and are thought to represent processing intermediates. The proAFP was unique to the transgenic plants and was absent in extracts taken prior to cold exposure. AFP levels increased over the first 48 h of cold incubation then remained stable. Since the -helix content of Type I AFP has been shown to decrease markedly at warmer temperatures, we postulate that Type I AFP stability in transgenic plants is dependent on its secondary structure.     

Identification of a two-domain antifreeze protein gene in Antarctic eelpout <Emphasis Type="Italic">Lycodichthys dearborni</Emphasis>

Type III antifreeze proteins (AFP III) in the Antarctic eelpout Lycodichthys dearborni contain at least two size variants—a 7-kDa protein family and a specific 14-kDa isoform composed of two 7-kDa domains linked in tandem. We report the characterization of a two-domain AFP III gene from L. dearborni, and propose that the two-domain AFP III gene arose from a single-domain AFP III gene through duplication and degeneration. AT-rich regions played an important role in the degeneration of the duplicated AFP III gene that resulted in the concatenation of two originally separated 7-kDa AFP-coding exons into a single gene. We also identified a pseudo-AFP III gene interrupted at an AT-rich coding region, supporting AT-rich regions as hotspots for DNA recombination in AFP III gene evolution. Interestingly, study of AFP III genes in the related Antarctic eelpout Pachycara brachycephalum showed absence of two- and multi-domain AFP III genes, indicating that modes of AFP III gene family evolution are specific within species. Nucleotide sequences have been deposited into NCBI Genbank under Accession Numbers: EU627165, EU627166.     

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.     

Solution Structures,Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.     

A natural variant of type I antifreeze protein with four ice-binding repeats is a particularly potent antifreeze.

A 4.3-kDa variant of Type I antifreeze protein (AFP9) was purified from winter flounder serum by size exclusion chromatography and reversed-phase HPLC. By the criteria of mass, amino acid composition, and N-terminal sequences of tryptic peptides, this variant is the posttranslationally modified product of the previously characterized AFP gene 21a. It has 52 amino acids and contains four 11-amino acid repeats, one more than the major serum AFP components. The larger protein is completely alpha-helical at 0 degree C, with a melting temperature of 18 degrees C. It is considerably more active as an antifreeze than the three-repeat winter flounder AFP and the four-repeat yellowtail flounder AFP, both on a molar and a mg/mL basis. Several structural features of the four-repeat winter flounder AFP, including its larger size, additional ice-binding residues, and differences in ice-binding motifs might contribute to its greater activity. Its abundance in flounder serum, together with its potency as an antifreeze, suggest that AFP9 makes a significant contribution to the overall freezing point depression of the host.     

Flounder antifreeze peptides increase the efficacy of cryosurgery

Type I antifreeze protein (AFP) from the winter flounder (Pseudopleuronectes americanus) was used as an adjuvant to cryosurgery of subcutaneous tumors of Dunning AT-1 rat prostate cells grown in Copenhagen rats. The cryosurgical procedure was performed with a commercially available cryosurgery device (CRYO-HIT, Galil Medical) with clinically relevant single- and double-freeze protocols. Injury was assessed with the alamar blue indicator of metabolic activity. The assay gave anomalous results when used to assess the extent of injury immediately following the procedure, underestimating the extent of injury. However, a double-freeze procedure with antifreeze protein present was found to give significantly better ablation than a double-freeze without AFP or a single-freeze with or without AFP.     

Type II fish antifreeze protein accumulation in transgenic tobacco does not confer frost resistance

Type II fish antifreeze protein (AFP) is active in both freezing point depression and the inhibition of ice recrystallization. This extensively disulfidebonded 14 kDa protein was targeted for accumulation in its pro and mature forms in the cytosol and apoplast of transgenic tobacco plants. Type II AFP gene constructs under control of a duplicate cauliflower mosaic virus 35S promoter, both with and without a native plant transit peptide sequence, were introduced into tobacco by Agrobacterium tumefaciensmediated transformation. AFP did not accumulate in the cytosol of transgenic plants, but active AFP was present as 2% of the total protein present in the apoplast. Plantproduced AFP was the same size as mature Type II AFP isolated from fish, and was comparable to wildtype AFP in thermal hysteresis activity and its effect on ice crystal morphology. Field trials conducted in late summer on R1 generation transgenic plants showed similar AFP accumulation in plants under field conditions at levels suitable for largescale production: but no difference in frost resistance was observed between transgenic and wildtype plants during the onset of early fall frosts.     

Activation of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Purified antifreeze proteins (AFPs) from the larvae of the beetle Dendroides canadensis do not produce the high levels of antifreeze activity seen in the hemolymph of overwintering larvae, even when the purified AFPs are assayed at very high concentrations. However, addition of certain proteins or agar (at concentrations sufficiently low that the gel state does not result) to the Dendroides AFP resulted in a 2–3-fold increase in activity. A 70-kDa protein with AFP-activating capabilities was purified from Dendroides larvae. Addition of this endogenous activator protein to a 4 mg·ml^-1 solution of AFP increased the activity of the AFPs to values comparable to those of the hemolymph of overwintering larvae. Data derived from a modified immunoblot technique demonstrate that the activators bind to the AFP, or vice versa. Formation of this association must allow the AFP to block ice crystal growth by binding to the surface of potential seed crystals in the normal fashion. However, because the AFP-activator complex is much larger than the AFP alone, the complex probably blocks a greater surface area of the crystal and is thus a more efficient antifreeze.Abbreviations AFP antifreeze protein - BSA bovine serum albumine - DEAE diethylaminoethyl - Ig immunoglubolin - LPIN lipoprotein ice nucleator - PIN protein ice nucleator - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - TH thermal hysteresis     

Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein.

To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase-directed expression vector. The 67-amino acid-long gene product differed from the natural AFP by inclusion of an N-terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis-trans proline isomerization from the C-terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity.     

Spatial expression patterns of skin-type antifreeze protein in winter flounder (Pseudopleuronectes americanus) epidermis following metamorphosis

Two isotypes of Type I antifreeze protein (AFP), the liver-type and the skin-type, have been described from adult winter flounder (Pseudopleuronectes americanus). Although the liver-type AFP has been well studied, the skin-type has just begun to be characterized. It appears to have a wide tissue distribution, be expressed constitutively, and the absence of a signal sequence suggests it is active intracellularly. The current study was designed to examine the onset of skin-type AFP expression during the thickening of the epidermis at metamorphosis from both the nucleic acid and protein levels. The epidermis appeared as a thin layer overlying a thickened dermis at metamorphosis and showed a gradual increase in thickness through the first fall and winter. The onset of skin-type antifreeze expression occurred in conjunction with this epidermal thickening. In situ hybridization and immunohistochemistry showed a distribution of mRNA and skin-type AFP specific for the epidermis and epidermal pavement cells. The AFP immunoproduct showed a distribution intimate with the pavement cell membrane and through the interstitial spaces. This distribution suggests that the AFP may be important in slowing ice crystal formation in these interstitial regions and thus reducing cellular damage due to osmotic imbalance.     

Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells

We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.     

Antifreeze proteins bind independently to ice.

It has been suggested that cooperative interactions between antifreeze proteins (AFPs) on the ice surfaces are required for complete inhibition of ice crystal growth. To test this hypothesis, a 7-kDa type III AFP was linked through its N-terminus to thioredoxin (12 kDa) or maltose-binding protein (42 kDa). The resultant 20-kDa and 50-kDa fusion proteins were larger in diameter than free AFP and thus precluded any extensive AFP-AFP contacts on the ice surface. Both fusion proteins were at least as active as free AFP at virtually all concentrations tested. By these criteria, AFPs function independently of each other and do not require specific intermolecular interactions to bind tightly to ice.     

A leucine-rich repeat protein of carrot that exhibits antifreeze activity

A gene encoding an antifreeze protein (AFP) was isolated from carrot (Daucus carota) using sequence information derived from the purified protein. The carrot AFP is highly similar to the polygalacturonase inhibitor protein (PGIP) family of apoplastic plant leucine-rich repeat (LRR) proteins. Expression of the AFP gene is rapidly induced by low temperatures. Furthermore, expression of the AFP gene in transgenic Arabidopsis thaliana plants leads to an accumulation of antifreeze activity. Our findings suggest that a new type of plant antifreeze protein has recently evolved from PGIPs.     

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     

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.