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Antifreeze proteins (AFPs) inhibit ice growth within fish and protect them from freezing in icy seawater. Alanine-rich, alpha-helical AFPs (type I) have independently (convergently) evolved in four branches of fishes, one of which is a subsection of the righteye flounders. The origin of this gene family has been elucidated by sequencing two loci from a starry flounder, Platichthys stellatus, collected off Vancouver Island, British Columbia. The first locus had two alleles that demonstrated the plasticity of the AFP gene family, one encoding 33 AFPs and the other allele only four. In the closely related Pacific halibut, this locus encodes multiple Gig2 (antiviral) proteins, but in the starry flounder, the Gig2 genes were found at a second locus due to a lineage-specific duplication event. An ancestral Gig2 gave rise to a 3-kDa “skin” AFP isoform, encoding three Ala-rich 11-a.a. repeats, that is expressed in skin and other peripheral tissues. Subsequent gene duplications, followed by internal duplications of the 11 a.a. repeat and the gain of a signal sequence, gave rise to circulating AFP isoforms. One of these, the “hyperactive” 32-kDa Maxi likely underwent a contraction to a shorter 3.3-kDa “liver” isoform. Present day starry flounders found in Pacific Rim coastal waters from California to Alaska show a positive correlation between latitude and AFP gene dosage, with the shorter allele being more prevalent at lower latitudes. This study conclusively demonstrates that the flounder AFP arose from the Gig2 gene, so it is evolutionarily unrelated to the three other classes of type I AFPs from non-flounders. Additionally, this gene arose and underwent amplification coincident with the onset of ocean cooling during the Cenozoic ice ages.

Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

The two main mammalian calpains, 1 and 2, are heterodimers of a large 80 kDa and a small 28 kDa subunit that together bind multiple calcium ions during enzyme activation. The main contact between the two subunits of these intracellular cysteine proteases is through a pairing of the fifth EF-hand of their C-terminal penta-EF-hand (PEF) domains. From modeling studies and observation of crystal structures, it is not obvious why these calpains form heterodimers with the small subunit rather than homodimers of the large subunit, as suggested for calpain 3 (p94). Therefore, we have used a differential tagging system to determine which of the other PEF domain-containing calpains form heterodimers and which form homodimers. His6-tagged PEF domains of calpains 1, 3, 9 and 13 were coexpressed with the PEF domain of the small subunit that had been tagged with an antifreeze protein. As predicted, the PEF domain of calpain 1 heterodimerized and that of calpain 3 formed a homodimer. The PEF domain of digestive tract-specific calpain 9 heterodimerized with the small subunit, and that of calpain 13, prevalent in lung and testis, was mainly found as a homodimer with a small amount of heterodimer. These results indicate whether recombinant production of a particular calpain requires coexpression of the small subunit, and whether this calpain is likely to be active in a small subunit knockout mouse. Furthermore, as the endogenous inhibitor calpastatin binds to PEF domains on the large and small subunit, it is less likely that the homodimeric calpains 3 and 13 with two active sites will bind or be silenced by calpastatin.

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 alpha-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 microg/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.

Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.

In the late stages of spermatogenesis, winter flounder produce a family of high molecular mass (80-200 kDa) basic nuclear proteins (HMrBNPs) that combine with the normal complement of histones to produce condensed sperm chromatin with an increased nucleosomal repeat length. The HMrBNPs have a biased amino-acid composition in which Arg, Ser, Lys and Pro are abundant because of their presence in many simple peptide repeats. The organization of these repeats was deduced by cDNA cloning. The predominant repeating units are related 26- and 30-amino-acid sequences that in turn are linked by 6-amino-acid spacers to form 58- and 62-amino-acid repeats. Subsets of these repeats are also present, such as a dispersed 20-amino-acid repeat and a tandem array of nine heptapeptides at the C-terminus. The HMrBNPs appear to have evolved from an extreme H1 variant that has an N-terminal tail of HMrBNP-like sequence linked to an H1 globular region. Based on sequences of the most abundant HMrBNP cDNAs, and the lack of hybridization between HMrBNP mRNAs and a DNA probe for the H1 globular region, the latter domain appears to have been lost during expansion and amplification of the HMrBNP-like repeats. Transcripts of the HMrBNP and H1 variant genes are present in testis RNAs only during the mid-spermatid stage of spermatogenesis, at the same time that HMrBNPs in their highly phosphorylated form first appear in the nucleus. Judging by the lack of a lag between HMrBNP mRNA synthesis and translation, the mRNAs for these highly basic proteins are not stored for any length of time. Instead, the deposition of HMrBNPs onto DNA, which coincides with the major reorganization and silencing of the chromatin, may be controlled by dephosphorylation.

We have analyzed the inheritance and expression of a line of transgenic salmon harboring the antifreeze protein gene from the winter flounder. The genomic clone 2A-7 coding for a major liver-type antifreeze protein gene (wflAFP-6) was integrated into the salmon genome. From a transgenic founder (#1469), an F3 generation was produced. In this study, southern blot analysis showed that only one copy of the antifreeze protein transgene was integrated into a unique site in F3 transgenic fish. The integration site was cloned and characterized. Northern analysis indicated that the antifreeze protein mRNA was only expressed in the liver and showed seasonal variation. All of the F3 offspring contained similar levels of the antifreeze protein precursor protein in the sera and the sera of these offspring showed a characteristic hexagonal ice crystal pattern indicating the presence of antifreeze activity. In addition, the antifreeze protein precursor protein level was found to vary with the season, being highest in the month of November and lowest in May. This study had demonstrated a tissue-specific and stable expression of the antifreeze protein transgene in the F3 generation of the transgenic salmon 1469 line.

The 9-kDa, Thr-, Ser-, and Cys-rich thermal hysteresis protein from spruce budworm (sbwTHP) is 10-30 times more effective than fish antifreeze proteins (AFPs) at depressing solution freezing points via ice-crystal growth inhibition. Since this insect protein is only available in microgram quantities from its natural source, recombinant sbwTHP was produced from inclusion bodies in Escherichia coli by a refolding protocol. Incompletely folded forms were removed during ion-exchange and reverse-phase chromatography, resulting in fully active sbwTHP that was indistinguishable in its properties from native sbwTHP. The antifreeze was completely inactivated by reduction, showed no reaction with sulfhydryl reagents, and was not inhibited by EDTA. All eight cysteine residues appear to be involved in disulfide bond formation. Tryptic cleavage and peptide analysis is consistent with linkages between the first and second cysteine residues, the third and fourth, fifth and eighth, and the sixth and seventh. NMR analysis confirmed that the fully folded form of sbwTHP was well structured and had a single conformation. Both NMR and CD spectra indicate the presence of extensive beta structure (70-80%) with little or no alpha helix. The protein maintains antifreeze activity over a broad range of pH values, and its conformation is independent of both temperature (over the range 0 degrees C to 20 degrees C), and the presence of 50% trifluoroethanol.

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.

Recombinant cystine-rich fish antifreeze protein (AFP) was produced by fall armyworm cells from a baculovirus expression vector containing sea raven AFP cDNA. The natural signal sequence encoded in the cDNA directed secretion of the antifreeze into the medium, from where it was recovered and purified to homogeneity. The M(r) of the exported protein (16k), as determined by SDS-PAGE, was larger than that (14k) of mature AFP isolated from sea raven serum. Sequencing of the recombinant AFP showed that it had a 17-amino-acid extension N-terminal to the 129-amino-acid mature AFP that began where signal peptide cleavage should occur according to current algorithms. Recombinant proAFP had antifreeze activity equivalent to that of the mature AFP, which indicates that the disulfide bonds were correctly formed and that the ice-binding site on the antifreeze is not sterically hindered by the 17-amino-acid N-terminal extension.

Three members, 11-3, F2 and 5a , of the type-I antifreeze protein (AFP) multigene family in winter flounder were sequenced. All three belong to the subset of AFP genes that are linked, but irregularly spaced, and show significant differences from the functional genes in tandem repeats. 11-3 and F2 appear to be pseudogenes. Their intron, 3′-exon and 3′-flanking DNAs are similar to those of other AFP genes, but their 5′-exon is either missing or extensively modified, and has stop codons present in all three reading frames. Based on a comparison of intron sequences of family members, 11-3/F2 may represent a residual progenitor AFP gene which was duplicated after reaching pseudogene status. The third gene, 5a, is remarkable in having a 3′-exon that encodes an exceptionally long, Ala-rich sequence that lacks any semblance of the 11-amino acid repeats found in 11-3, F2 and functional AFP genes. 5a might also be a pseudogene, because its presumed TATA box appears to have mutated.

The recent discovery of a large hyperactive antifreeze protein in the blood plasma of winter flounder has helped explain why this fish does not freeze in icy seawater. The previously known, smaller and much less active type I antifreeze proteins cannot by themselves protect the flounder down to the freezing point of seawater. The relationship between the large and small antifreezes has yet to be established, but they do share alanine-richness (> 60%) and extensive alpha-helicity. Here we have examined two other righteye flounder species for the presence of the hyperactive antifreeze, which may have escaped prior detection because of its lability. Such a protein is indeed present in the yellowtail flounder judging by its size, amino acid composition and N-terminal sequence, along with the previously characterized type I antifreeze proteins. An ortholog is also present in American plaice based on the above criteria and its high specific antifreeze activity. This protein was purified and shown to be almost fully alpha-helical, highly asymmetrical, and susceptible to denaturation at room temperature. It is the only detectable antifreeze protein in the blood plasma of the American plaice. Because this species appears to lack the smaller type I antifreeze proteins, the latter may have evolved by descent from the larger antifreeze.

The circulating Type II antifreeze protein (AFP) in sea raven is 129 amino acids (aa) long (14 kDa) and is derived from an initial 163 aa translation product that is synthesised in the liver. Signal peptide cleavage algorithms, as well as transgenic expression studies in fall armyworm cells, predict the formation of a 146 aa (16 kDa) proprotein intermediate. A protein of this size that cross-reacted with anti-sea raven AFP antibody was detected in sea raven serum using phosphate/urea SDS-PAGE, and was purified by size-exclusion chromatography and reverse-phase HPLC. N-terminal sequencing and mass spectrometry identified the protein as the predicted proAFP, and immunoblotting suggested that it is the predominant form present in liver. These results are consistent with production and storage of a proAFP intermediate in the liver, and its subsequent processing to mature AFP during or soon after its release into the circulation.