Various microbes, including fungi and bacteria, that live in cold environments produce ice‐binding proteins (IBPs) that protect them from freezing. Ascomycota and Basidiomycota are two major phyla of fungi, and Antarctomyces psychrotrophicus is currently designated as the sole ascomycete that produces IBP (AnpIBP). However, its complete amino acid sequence, ice‐binding property, and evolutionary history have not yet been clarified. Here, we determined the peptide sequences of three new AnpIBP isoforms by total cDNA analysis and compared them with those of other microbial IBPs. The AnpIBP isoforms and ascomycete‐putative IBPs were found to be phylogenetically close to the bacterial ones but far from the basidiomycete ones, which is supported by the higher sequence identities to bacterial IBPs than basidiomycete IBPs, although ascomycetes are phylogenetically distant from bacteria. In addition, two of the isoforms of AnpIBP share low sequence identity and are not close in the phylogenetic tree. It is hence presumable that these two AnpIBP isoforms were independently acquired from different bacteria through horizontal gene transfer (HGT), which implies that ascomycetes and bacteria frequently exchange their IBP genes. The non‐colligative freezing‐point depression ability of AnpIBP was not very high, whereas it exhibited significant abilities of ice recrystallization inhibition, ice shaping, and cryo‐protection against freeze–thaw cycles even at submicromolar concentrations. These results suggest that HGT is crucial for the cold‐adaptive evolution of ascomycetes, and their IBPs offer freeze resistance to organisms to enable them to inhabit the icy environments of Antarctica.Databases: Nucleotide sequence data are available in the DDBJ database under the accession numbers LC378707, LC378707, LC378707 for AnpIBP1a, AnpIBP1b, AnpIBP2, respectively.

Polypentagonal water networks were recently observed in a protein capable of binding to ice crystals, or ice-binding protein (IBP). To examine such water networks and clarify their role in ice-binding, we determined X-ray crystal structures of a 65-residue defective isoform of a Zoarcidae-derived IBP (wild type, WT) and its five single mutants (A20L, A20G, A20T, A20V, and A20I). Polypentagonal water networks composed of ∼50 semiclathrate waters were observed solely on the strongest A20I mutant, which appeared to include a tetrahedral water cluster exhibiting a perfect position match to the $\left(10\overline{1}0\right)$ first prism plane of a single ice crystal. Inclusion of another symmetrical water cluster in the polypentagonal network showed a perfect complementarity to the waters constructing the $\left(20\overline{2}1\right)$ pyramidal ice plane. The order of ice-binding strength was A20L < A20G < WT < A20T < A20V < A20I, where the top three mutants capable of binding to the first prism and the pyramidal ice planes commonly contained a bifurcated γ-CH₃ group. These results suggest that a fine-tuning of the surface of Zoarcidae-derived IBP assisted by a side-chain group regulates the holding property of its polypentagonal water network, the function of which is to freeze the host protein to specific ice planes. Significance This study expands our knowledge of protein hydration, which is highly related to the macromolecular antifreeze property of proteins. We examined a polypentagonal network formation of waters for a series of artificial variants of a 65-residue ice-binding protein. The polypentagonal waters were created solely on the surface of an activity-improved variant, which appeared to contain two sets of water clusters exhibiting a perfect position match to the waters constructing the first prism and pyramidal ice planes. These data suggest that a minute structural change in a protein organizes the surface waters into a polypentagonal arrangement, which merges with the intrinsically disordered ice surface and freezes to specific ice crystal planes.

The concentration of a protein is highly related to its biochemical properties, and is a key determinant for its biotechnological applications. Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are structurally diverse macromolecules that are capable of binding to embryonic ice crystals below 0 °C, making them useful as protectants of ice-block formation. In this study, we examined the maximal solubility of native AFP I–III and AFGP with distilled water, and evaluated concentration dependence of their ice-binding property. Approximately 400 mg/mL (AFP I), 200 mg/mL (AFP II), 100 mg/mL (AFP III), and >1800 mg/mL (AFGP) of the maximal solubility were estimated, and among them AFGP’s solubility is much higher compared with that of ordinary proteins, such as serum albumin (~500 mg/mL). The samples also exhibited unexpectedly high thermal hysteresis values (2–3 °C) at 50–200 mg/mL. Furthermore, the analysis of fluorescence-based ice plane affinity showed that AFP II binds to multiple ice planes in a concentration-dependent manner, for which an oligomerization mechanism was hypothesized. The difference of concentration dependence between AFPs and AFGPs may provide a new clue to help us understand the ice-binding function of these proteins.

Hydration is crucial for a function and a ligand recognition of a protein. The hydration shell constructed on an antifreeze protein (AFP) contains many organized waters, through which AFP is thought to bind to specific ice crystal planes. For a Ca²⁺-dependent species of AFP, however, it has not been clarified how 1 mol of Ca²⁺-binding is related with the hydration and the ice-binding ability. Here we determined the X-ray crystal structure of a Ca²⁺-dependent AFP (jsAFP) from Japanese smelt, Hypomesus nipponensis, in both Ca²⁺-bound and -free states. Their overall structures were closely similar (Root mean square deviation (RMSD) of Cα = 0.31 Å), while they exhibited a significant difference around their Ca²⁺-binding site. Firstly, the side-chains of four of the five Ca²⁺-binding residues (Q92, D94 E99, D113, and D114) were oriented to be suitable for ice binding only in the Ca²⁺-bound state. Second, a Ca²⁺-binding loop consisting of a segment D94–E99 becomes less flexible by the Ca²⁺-binding. Third, the Ca²⁺-binding induces a generation of ice-like clathrate waters around the Ca²⁺-binding site, which show a perfect position-match to the waters constructing the first prism plane of a single ice crystal. These results suggest that generation of ice-like clathrate waters induced by Ca²⁺-binding enables the ice-binding of this protein.

Cells for therapeutic use are often preserved at +4 °C, and the storage period is generally limited to 2–3 days. Here, we report that the survival rate (%) of mammalian cells is improved to 10–20 days when they are preserved with a subzero supercooled solution containing the antifreeze protein (AFP), for which an ability to stabilize both supercooled water and cell membrane integrity has been postulated. We chose adherent rat insulinoma (RIN-5F) cells as the preservation target, which were immersed into −5 °C-, −2 °C-, or +4 °C-chilled “unfrozen” solution of Euro-Collins or University of Washington (UW) containing the AFP sample obtained from insect or fish. Our results show that the survival rate of the cells preserved with the solution containing insect AFP was always higher than that of the fish AFP solution. A combination of the −5 °C-supercooling and insect AFP gave the best preservation result, namely, UW solution containing insect AFP kept 53% of the cells alive, even after 20 days of preservation at −5 °C. The insect AFP locates highly organized ice-like waters on its molecular surface. Such waters may bind to semiclathrate waters constructing both embryonic ice crystals and a membrane–water interface in the supercooled solution, thereby protecting the cells from damage due to chilling.

Beetle hyperactive antifreeze protein (AFP) has a unique ability to maintain a supercooling state of its body fluids, however, less is known about its origination. Here, we found that a popular stag beetle Dorcus hopei binodulosus (Dhb) synthesizes at least 6 isoforms of hyperactive AFP (DhbAFP). Cold-acclimated Dhb larvae tolerated −5 °C chilled storage for 24 h and fully recovered after warming, suggesting that DhbAFP facilitates overwintering of this beetle. A DhbAFP isoform (~10 kDa) appeared to consist of 6−8 tandem repeats of a 12-residue consensus sequence (TCTxSxNCxxAx), which exhibited 3 °C of high freezing point depression and the ability of binding to an entire surface of a single ice crystal. Significantly, these properties as well as DNA sequences including the untranslated region, signal peptide region, and an AFP-encoding region of Dhb are highly similar to those identified for a known hyperactive AFP (TmAFP) from the beetle Tenebrio molitor (Tm). Progenitor of Dhb and Tm was branched off approximately 300 million years ago, so no known evolution mechanism hardly explains the retainment of the DNA sequence for such a lo­ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.