The ice-binding site of antifreeze protein irreversibly binds to cell surface for its hypothermic protective function.

Antifreeze proteins (AFPs) are multifunctional polypeptides that adsorb onto ice crystals to inhibit their growth and onto cells to protect them from nonfreezing hypothermic damage. However, the mechanism by which AFP exerts its hypothermic cell protective (HCP) function remains uncertain. Here, we assessed the HCP function of three types of fish-derived AFPs (type I, II, and III AFPs) against human T-lymphoblastic lymphoma by measuring the survival rate (%) of the cells after preservation at 4 °C for 24 h. All AFPs improved the survival rate in a concentration-dependent manner, although the HCP efficiency was inferior for type III AFP compared to other AFPs. In addition, after point mutations were introduced into the ice-binding site (IBS) of a type III AFP, HCP activity was dramatically increased, suggesting that the IBS of AFP is involved in cell adsorption. Significantly, high HCP activity was observed for a mutant that exhibited poorer antifreeze activity, indicating that AFP exerts HCP- and ice-binding functions through a different mechanism. We next incubated the cells in an AFP-containing solution, replaced it with pure EC solution, and then preserved the cells, showing that no significant reduction in the cell survival rate occurred for type I and II AFPs even after replacement. Thus, these AFPs irreversibly bind to the cells at 4 °C, and only tightly adsorbed AFP molecules contribute towards the cell-protection function.

Regulating Antifreeze Activity through Water: Latent Functions of the Sugars of Antifreeze Glycoprotein Revealed by Total Chemical Synthesis.

Antifreeze glycoprotein (AFGP), which inhibits the freezing of water, is highly O-glycosylated with a disaccharide, d-Galß1-3-d-GalNAcα (GalGalNAc). To elucidate the function of the sugar residues for antifreeze activity at the molecular level, we conducted a total chemical synthesis of partially sugar deleted AFGP derivatives, and unnatural forms of AFGPs incorporating glucose (Glc)-type sugars instead of galactose (Gal)-type sugars. These elaborated AFGP derivatives demonstrated that the stereochemistry of each sugar residue on AFGPs precisely correlates with the antifreeze activity. A hydrogen-deuterium exchange experiment using synthetic AFGPs revealed a different dynamic behavior of water around sugar residues depending on the sugar structures. These results indicate that sugar residues on AFGP form a unique dynamic water phase that disturbs the absorbance of water molecules onto the ice surface, thereby inhibiting freezing.

A mutation to a fish ice-binding protein synthesized in transgenic Caenorhabditis elegans modulates its cold tolerance.

A cryoprotectant known as ice-binding protein (IBP) is thought to facilitate the cold survival of plants, insects, and fungi. Here, we prepared a genetically modified Caenorhabditis elegans strain to synthesize fish-derived IBPs in its body wall muscles and examined whether the antifreeze activity modification of this IBP by point mutation affects the cold tolerance of this worm. We chose a 65-residue IBP identified from notched-fin eelpout, for which the replacement of the 20th alanine residue (A20) modifies its antifreeze activity. These mutant proteins are denoted A20L, A20G, A20T, A20V, and A20I along with the wild-type (WT) protein. We evaluated the survival rate (%) of the transgenic C. elegans that synthesized each IBP mutant following 24 h of preservation at -5, +2, and +5 °C. Significantly, a dramatic improvement in the survival rate was detected for the worms synthesizing the activity-enhanced mutants (A20T and A20I), especially at +2 °C. In contrast, the rate was not improved by the expression of the defective mutants (A20L, A20G, WT and A20V). The survival rate (%) probably correlates with the antifreeze activity of the IBP. These data suggest that IBP protects the cell membrane by employing its ice-binding mechanism, which ultimately improves the cold tolerance of an IBP-containing animal.

Visualizing Intramolecular Dynamics of Membrane Proteins.

Membrane proteins play important roles in biological functions, with accompanying allosteric structure changes. Understanding intramolecular dynamics helps elucidate catalytic mechanisms and develop new drugs. In contrast to the various technologies for structural analysis, methods for analyzing intramolecular dynamics are limited. Single-molecule measurements using optical microscopy have been widely used for kinetic analysis. Recently, improvements in detectors and image analysis technology have made it possible to use single-molecule determination methods using X-rays and electron beams, such as diffracted X-ray tracking (DXT), X-ray free electron laser (XFEL) imaging, and cryo-electron microscopy (cryo-EM). High-speed atomic force microscopy (HS-AFM) is a scanning probe microscope that can capture the structural dynamics of biomolecules in real time at the single-molecule level. Time-resolved techniques also facilitate an understanding of real-time intramolecular processes during chemical reactions. In this review, recent advances in membrane protein dynamics visualization techniques were presented.

Polypentagonal ice-like water networks emerge solely in an activity-improved variant of ice-binding protein.

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 [Formula: see text] 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 [Formula: see text] 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.

Discovery of Hyperactive Antifreeze Protein from Phylogenetically Distant Beetles Questions Its Evolutionary Origin.

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 lo-ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.

Subzero Nonfreezing Hypothermia with Insect Antifreeze Protein Dramatically Improves Survival Rate of Mammalian Cells.

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.

Presence of a basic secretory protein in xylem sap and shoots of poplar in winter and its physicochemical activities against winter environmental conditions.

XSP25, previously shown to be the most abundant hydrophilic protein in xylem sap of Populus nigra in winter, belongs to a secretory protein family in which the arrangement of basic and acidic amino acids is conserved between dicotyledonous and monocotyledonous species. Its gene expression was observed at the same level in roots and shoots under long-day conditions, but highly induced under short-day conditions and at low temperatures in roots, especially in endodermis and xylem parenchyma in the root hair region of Populus trichocarpa, and its protein level was high in dormant buds, but not in roots or branches. Addition of recombinant PtXSP25 protein mitigated the denaturation of lactate dehydrogenase by drying, but showed only a slight effect on that caused by freeze-thaw cycling. Recombinant PtXSP25 protein also showed ice recrystallization inhibition activity to reduce the size of ice crystals, but had no antifreezing activity. We suggest that PtXSP25 protein produced in shoots and/or in roots under short-day conditions and at non-freezing low temperatures followed by translocation via xylem sap to shoot apoplast may protect the integrity of the plasma membrane and cell wall functions from freezing and drying damage in winter environmental conditions.

Applications of Antifreeze Proteins: Practical Use of the Quality Products from Japanese Fishes.

Numerous embryonic ice crystals are generated in water at the moment of freezing. These crystals grow and merge together to form an ice block that can be generally observed. Antifreeze protein (AFP) is capable of binding to the embryonic ice crystals, inhibiting such an ice block formation. Fish-derived AFP additionally binds to membrane lipid bilayers to prolong the lifetime of cells. These unique abilities of AFP have been studied extensively for the development of advanced techniques, such as ice recrystallization inhibitors, freeze-tolerant gels, cell preservation fluids, and high-porosity ceramics, for which mass-preparation method of the quality product of AFP utilizing fish muscle homogenates made a significant contribution. In this chapter, we present both fundamental and advanced information of fish AFPs that have been especially discovered from mid-latitude sea area, which will provide a hint to develop more advanced techniques applicable in both medical and industrial fields.

Total Synthesis of O-GalNAcylated Antifreeze Glycoprotein using the Switchable Reactivity of Peptidyl-N-pivaloylguanidine.

Antifreeze glycoprotein (AFGP) is an O-glycoprotein that displays antifreeze activity through depression of the freezing point of water. GalNAc is a core sugar structure of AFGP, and contributes to induce antifreeze activity of this glycoprotein. However, the general functional role that this sugar plays at the molecular level is still unknown. To elucidate this, it is essential to determine the relationship between structure and activity of O-GalNAcylated AFGP using homogeneous glycoproteins. Thus, the total synthesis of homogeneous O-GalNAcylated AFGP was conducted by using a unique peptide derivative: peptidyl-N-pivaloylguanidine. It was found that peptidyl-N-pivaloylguanidine is an "unreactive" peptide in peptide coupling reactions but is interconvertible with a "reactive" peptide-α-thioester by means of a simple treatment under buffer condition at pH=7 to 8. The unique switchable reactivity of peptidyl-N-pivaloylguanidine enabled an efficient sequential peptide coupling strategy. By using this strategy, various lengths of homogeneous O-GalNAcylated AFGP were synthesized, including one that was 120 amino acids in length, with 40 O-GalNAcylation sites. The structural analysis by circular dichroism spectroscopy and evaluation of the antifreeze activity of the synthetic AFGP(GalNAc)s revealed that the simple O-glycosylation with GalNAc is essential for both structural and functional basis of AFGP to exhibit antifreeze activity.

Hydrophobic ice-binding sites confer hyperactivity of an antifreeze protein from a snow mold fungus.

Snow mold fungus, Typhula ishikariensis, secretes seven antifreeze protein isoforms (denoted TisAFPs) that assist in the survival of the mold under snow cover. Here, the X-ray crystal structure of a hyperactive isoform, TisAFP8, at 1.0 Å resolution is presented. TisAFP8 folds into a right-handed ß-helix accompanied with a long α-helix insertion. TisAFP8 exhibited significantly high antifreeze activity that is comparable with other hyperactive AFPs, despite its close structural and sequence similarity with the moderately active isoform TisAFP6. A series of mutations introduced into the putative ice-binding sites (IBSs) in the ß-sheet and adjacent loop region reduced antifreeze activity. A double-mutant A20T/A212S, which comprises a hydrophobic patch between the ß-sheet and loop region, caused the greatest depression of antifreeze activity of 75%, when compared with that of the wild-type protein. This shows that the loop region is involved in ice binding and hydrophobic residues play crucial functional roles. Additionally, bound waters around the ß-sheet and loop region IBSs were organized into an ice-like network and can be divided into two groups that appear to mediate separately TisAFP and ice. The docking model of TisAFP8 with the basal plane via its loop region IBS reveals a better shape complementarity than that of TisAFP6. In conclusion, we present new insights into the ice-binding mechanism of TisAFP8 by showing that a higher hydrophobicity and better shape complementarity of its IBSs, especially the loop region, may render TisAFP8 hyperactive to ice binding.

Prolonging hypothermic storage (4 C) of bovine embryos with fish antifreeze protein.

Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24-48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP-embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.

Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation.

Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular ß-helix with six loops of 18 or more residues that lies alongside an α-helix. ß-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the ß-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of ß-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.

Identification of a novel LEA protein involved in freezing tolerance in wheat.

Late embryogenesis abundant (LEA) proteins are a family of hyper-hydrophilic proteins that accumulate in response to cellular dehydration. Originally identified as plant proteins associated with seed desiccation tolerance, LEA proteins have been identified in a wide range of organisms such as invertebrates and microorganisms. LEA proteins are thought to protect proteins and biomembranes under water-deficit conditions. Here, we characterized WCI16, a wheat (Triticum aestivum) protein that belongs to a class of plant proteins of unknown function, and provide evidence that WCI16 shares common features with LEA proteins. WCI16 was induced during cold acclimation in winter wheat. Based on its amino acid sequence, WCI16 is highly hydrophilic, like LEA proteins, despite having no significant sequence similarity to any of the known classes of LEA proteins. Recombinant WCI16 protein was soluble after boiling, and (1)H-nuclear magnetic resonance (NMR) spectroscopy revealed that the structure of WCI16 is random and has no hydrophobic regions. WCI16 exhibited in vitro cryoprotection of the freeze-labile enzyme l-lactate dehydrogenase as well as double-stranded DNA binding activity, suggesting that WCI16 may protect both proteins and DNA during environmental stresses. The biological relevance of these activities was supported by the subcellular localization of a green fluorescent protein (GFP)-fused WCI16 protein in the nucleus and cytoplasm. Heterologous expression of WCI16 in Arabidopsis (Arabidopsis thaliana) plants conferred enhanced freezing tolerance. Taken together, our results indicate that WCI16 represents a novel class of LEA proteins and is involved in freezing tolerance.

Annealing condition influences thermal hysteresis of fungal type ice-binding proteins.

The Antarctic sea ice diatom Navicular glaciei produced ice-binding protein (NagIBP) that is similar to the antifreeze protein (TisAFP) from snow mold Typhula ishikariensis. In the thermal hysteresis range of NagIBP, ice growth was completely inhibited. At the freezing point, the ice grew in a burst to 6 direction perdicular to the c-axis of ice crystal. This burst pattern is similar to TisAFP and other hyperactive AFPs. The thermal hysteresis of NagIBP and TisAFP could be increased by decreasing a cooling rate to allow more time for the proteins to bind ice. This suggests the possible second binding of proteins occurs on the ice surface, which might increase the hysteresises to a sufficient level to prevent freezing of the brine pockets which habitat of N. glaciei. The secondary ice binding was described as that after AFP molecules bind onto the flat ice plane irreversibly, which was based on adsorption-inhibition mechanism model at the ice-water interface, convex ice front was formed and overgrew during normal TH measurement (no annealing) until uncontrolled growth at the nonequilibrium freezing point. The results suggested that NagIBP is a hyperactive AFP that is expressed for freezing avoidance.

Microwave-assisted solid-phase synthesis of antifreeze glycopeptides.

Microwave-assisted solid-phase synthesis allows for the rapid and large-scale preparation and structure-activity characterization of tandem repeating glycopeptides, namely monodispersed synthetic antifreeze glycopeptides (syAFGPs, H-[Ala-Thr(Galß1,3GalNAcα1→)-Ala]n -OH, n=2-6). By employing novel AFGP analogues, we have demonstrated that of the monodispersed syAFGPn (n=2-6, degree of polymerization, DP=2-6, Mw =1257-3690 Da), syAFGP5 (DP=5, Mw =3082 Da) and syAFGP6 (DP=6, Mw =3690 Da) exhibit the ability to form typical hexagonal bipyramidal ice crystals and satisfactory thermal hysteresis activity. Structural characterization by NMR and CD spectroscopy revealed that syAFGP6 forms a typical poly-L-proline type II helix-like structure in aqueous solution whereas enzymatic modification by sialic acid of the residues at the C-3 positions of the nonreducing Gal residues disturbs this conformation and eliminates the antifreeze activity.

The effect of ice-binding proteins on the cryopreservation of Caenorhabditis elegans.

Ice-binding proteins (IBPs) are capable of binding ice crystals and inhibiting their growth. IBPs have also been reported to stabilize cell membranes under non-freezing conditions. The effects of IBPs help to reduce cold- and freezing-induced damage to cells and tissues in cryopreservation. Here, we examined whether certain IBPs, namely, fish NfeIBP6 and NfeIBP8 and fungal AnpIBP1a N55D (AnpIBP), improve the recovery rate of the nematode Caenorhabditis elegans after a deep cryopreservation at -80°C. The expression of fungus-derived AnpIBP in C. elegans significantly improved its recovery rate. This result provides useful information to establish a cryopreservation technique for long-term storage using IBP molecules.

Adsorption of ice-binding proteins onto whole ice crystal surfaces does not necessarily confer a high thermal hysteresis activity.

Many psychrophilic microorganisms synthesize ice-binding proteins (IBPs) to survive the cold. The functions of IBPs are evaluated by the effect of the proteins on the nonequilibrium water freezing-point depression, which is called "thermal hysteresis (TH)", and the inhibitory effect of the proteins on the growth of larger ice crystals, which is called "ice recrystallization inhibition (IRI)". To obtain mechanical insight into the two activities, we developed a modified method of ice affinity purification and extracted two new IBP isoforms from Psychromyces glacialis, an Arctic glacier fungus. One isoform was found to be an approximately 25 kDa protein (PsgIBP_S), while the other is a 28 kDa larger protein (PsgIBP_L) that forms an intermolecular dimer. Their TH activities were less than 1 °C at millimolar concentrations, implying that both isoforms are moderately active but not hyperactive IBP species. It further appeared that both isoforms exhibit high IRI activity even at submicromolar concentrations. Furthermore, the isoforms can bind to the whole surface of a hemispherical single ice crystal, although such ice-binding was generally observed for hyperactive IBP species. These results suggest that the binding ability of IBPs to whole ice crystal surfaces is deficient for hyperactivity but is crucial for significant IRI activity.

Dynamic motions of ice-binding proteins in living Caenorhabditis elegans using diffracted X-ray blinking and tracking.

The dynamic properties of protein molecules are involved in the relationship between their structure and function. Time-resolved X-ray observation enables capturing the structures of biomolecules with picometre-scale precision. However, this technique has yet to be implemented in living animals. Here, we examined diffracted X-ray blinking (DXB) and diffracted X-ray tracking (DXT) to observe the dynamics of a protein located on intestinal cells in adult Caenorhabditis elegans. This in vivo tissue-specific DXB was examined at temperatures from 20 °C to -10 °C for a recombinant ice-binding protein from Antarctomyces psychrotrophicus (AnpIBP) connected with the cells through a transmembrane CD4 protein equipped with a glycine-serine linker. AnpIBP inhibits ice growth at subzero temperatures by binding to ice crystals. We found that the rotational motion of AnpIBP decreases at -10 °C. In contrast, the motion of the AnpIBP mutant, which has a defective ice-binding ability, did not decrease at -10 °C. The twisting and tilting motional speeds of AnpIBPs measured above 5 °C by DXT were always higher than those of the defective AnpIBP mutant. These results suggest that wild-type AnpIBP is highly mobile in solution, and it is halted at subzero temperatures through ice binding. DXB and DXT allow for exploring protein behaviour in live animals with subnano resolution precision.

Synthetic study and structural analysis of the antifreeze agent xylomannan from Upis ceramboides.

The novel antifreeze factor, xylomannan, first isolated from the freeze-tolerant Alaskan beetle Upis ceramboides , demonstrates a high degree of thermal hysteresis, comparable to that of the most active insect antifreeze proteins. Although the presence of a lipid component in this factor has not yet been verified, it has been proposed that the glycan backbone consists of a ß-D-mannopyranosyl-(1→4)-ß-D-xylopyranose-disaccharide-repeating structure according to MS and NMR analyses. In this contribution, we report the stereoselective synthesis of the tetrasaccharide ß-D-mannopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→4)-ß-D-mannopyranosyl-(1→4)-D-xylopyranoside, a structural component of xylomannan. Our synthesis features the use of 2-naphthylmethyl (NAP)-ether-mediated intramolecular aglycon delivery (IAD) as the key reaction in obtaining ß-mannopyranoside stereoselectively. Various donors for NAP-IAD were tested to determine the most suitable for the purposes of this synthesis. Fragment coupling between a disaccharyl fluoride and a disaccharide acceptor obtained from a common ß-D-mannopyranosyl-(1→4)-ß-D-xylopyranoside derivative was successfully carried out to afford the desired tetrasaccharide in the presence of Cp(2)HfCl(2)-AgClO(4). Structural analysis of the resulting synthetic tetrasaccharide using NMR techniques and molecular modeling was performed in order to demonstrate the presence of the proposed xylomannan linkages in this molecule.