Recent Advances in Antifreeze Peptide Preparation: A Review.

Antifreeze agents play a critical role in various fields including tissue engineering, gene therapy, therapeutic protein production, and transplantation. Commonly used antifreeze agents such as DMSO and other organic substances are known to have cytotoxic effects. Antifreeze proteins sourced from cold-adapted organisms offer a promising solution by inhibiting ice crystal formation; however, their effectiveness is hindered by a dynamic ice-shaping (DIS) effect and thermal hysteresis (TH) properties. In response to these limitations, antifreeze peptides (AFPs) have been developed as alternatives to antifreeze proteins, providing similar antifreeze properties without the associated drawbacks. This review explores the methods for acquiring AFPs, with a particular emphasis on chemical synthesis. It aims to offer valuable insights and practical implications to drive the realm of sub-zero storage.

Globular Antifreeze Protein-Inspired Nanoparticle-Based Large-Scale T-Cell Cryoprotection System for Lymphoma Immunotherapy.

Large-scale biosafe T-cell cryopreservation is required to bring T-cell therapies to the market, but it remains challenging due to the cytotoxicity of common cryoprotectants [e.g., dimethyl sulfoxide (DMSO)] and unavoidable ice injuries to cells. Herein, inspired by natural globular antifreeze proteins, we establish a biocompatible zwitterionic magnetic nanoparticle (ZMNP)-based cryoprotection system, achieving large-scale cryopreservation of T cells for lymphoma immunotherapy. ZMNPs could form a globular hydration shell to inhibit water molecule aggregation as well as ice growth, and the surficial hydration strength-antifreeze performance relationship of ZMNPs was investigated. During the thawing process, ZMNPs possessed a magnetic field-mediated nanowarming property that enabled rapid heating and also facilitated easy magnetic separation for cell recovery. These combined effects resulted in a high post-thaw viability (>80%) of large-scale T-cell cryopreservation (20 mL). Notably, post-thaw T cells exhibited similar transcript profiles to fresh cells, while up- or downregulation of 1050 genes was found in the DMSO group. In a mouse E.G7-OVA lymphoma model, ZMNP-system-cryopreserved T cells achieved a tumor suppression rate of 77.5%, twice as high as the DMSO group. This work holds great promise for the application of advanced cryopreservation techniques in the development of therapeutic cellular products.

Design of eco-friendly antifreeze peptides as novel inhibitors of gas-hydration kinetics.

In this study, peptides designed using fragments of an antifreeze protein (AFP) from the freeze-tolerant insect Tenebrio molitor, TmAFP, were evaluated as inhibitors of clathrate hydrate formation. It was found that these peptides exhibit inhibitory effects by both direct and indirect mechanisms. The direct mechanism involves the displacement of methane molecules by hydrophobic methyl groups from threonine residues, preventing their diffusion to the hydrate surface. The indirect mechanism is characterized by the formation of cylindrical gas bubbles, the morphology of which reduces the pressure difference at the bubble interface, thereby slowing methane transport. The transfer of methane to the hydrate interface is primarily dominated by gas bubbles in the presence of antifreeze peptides. Spherical bubbles facilitate methane migration and potentially accelerate hydrate formation; conversely, the promotion of a cylindrical bubble morphology by two of the designed systems was found to mitigate this effect, leading to slower methane transport and reduced hydrate growth. These findings provide valuable guidance for the design of effective peptide-based inhibitors of natural-gas hydrate formation with potential applications in the energy and environmental sectors.

Assessment of the impact of whey protein hydrolysate on myofibrillar proteins in surimi during repeated freeze-thaw cycles: Quality enhancement and antifreeze potential.

The quality of surimi, widely used in processed seafood, is compromised by freeze-thaw cycles, leading to protein denaturation and oxidative degradation. The objective of this study is to explore the effects of adding natural whey peptide hydrolysate (WPH) on the myofibrillar proteins of repeatedly freeze-thawed surimi. Results indicated surimi treated with 15% WPH exhibited only a 128% increase in surface hydrophobicity and a maximum peroxide value of 7.84 µg/kg, significantly lower than the control group. Additionally, salt-soluble protein content, emulsification activity, and stability decreased with the increase in freeze-thaw cycles. With a 15% WPH offering the most significant protective effect, evidenced by reductions of only 25.02%, 42.52% and 37.02% in salt-soluble protein content, emulsification activity, and stability, respectively. These outcomes demonstrate that WPH effectively reduces protein denaturation during repeated freeze-thaw processes. Future research should explore the molecular mechanisms underlying WPH's protective effects and evaluate their applicability in other food systems.

Glycosylated Polyhydroxyproline Is a Potent Antifreeze Molecule.

Molecules that inhibit the growth of ice crystals are highly desirable for applications in building materials, foods, and agriculture. Antifreezes are particularly essential in biomedicine for tissue banking, yet molecules currently in use have known toxic effects. Antifreeze glycoproteins have evolved naturally in polar fish species living in subzero climates, but practical issues with collection and purification have limited their commercial use. Here, we present a synthetic strategy using polymerization of amino acid N-carboxyanhydrides to produce polypeptide mimics of these potent natural antifreeze proteins. We investigated a set of mimics with varied structural properties and identified a glycopolypeptide with potent ice recrystallization inhibition properties. We optimized for molecular weight, characterized their conformations, and verified their cytocompatibility in a human cell line. Overall, we present a material that will have broad applications as a biocompatible antifreeze.

Synergistic Effect of Hyperactive Antifreeze Protein on Inhibition of Gas-Hydrate Growth by Hydrophobic and Hydrophilic Groups.

Antifreeze proteins (AFPs) are biodegradable inhibitors that effectively prevent the formation of natural gas hydrates that block pipelines. In this study, molecular dynamics simulations were employed to establish a kinetic model of the hyperactive insect antifreeze protein (Tenebrio molitor, TmAFP) and its mutants to inhibit the growth of sI natural methane hydrate. Simulations revealed that the hydrophobic and hydrophilic groups of threonine (Thr) residues at hydrate-binding sites played a synergistic role in binding hydrates. The hydrophobic groups anchored TmAFP to the hydrate surface through residues Thr39-Thr65 by migrating pendant hydrophobic methyl groups to the hydrate semicages. The hydrophilic groups stabilized TmAFP by hydrogen bonding with water molecules and integrating them into a quasi-hydrate structure, which more effectively inhibited hydrate growth. The results suggest that the hydrate growth inhibition is attributed to both the shape complementarity and the flexibility of binding residues. The synergy between hydrophobic and hydrophilic groups provides guidance for the design of more effective hydrate inhibitors.

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.

Polyproline type II helical antifreeze proteins are widespread in Collembola and likely originated over 400 million years ago in the Ordovician Period.

Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, has not yet been adequately assessed. Here, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. Several methods were used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these AFPs had a high glycine content and were predicted to have the same polyproline type II helical bundle fold, a fold unique to Collembola. These Hexapods arose in the Ordovician Period with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish which arose independently during the Cenozoic Ice Age beginning ~ 30 million years ago.

Protein engineering of antifreeze proteins reveals that their activity scales with the area of the ice-binding site.

Antifreeze proteins (AFPs) protect organisms from freezing by binding to ice crystals to prevent their growth. Here, we have investigated how the area of an AFP's ice-binding site (IBS) changes its antifreeze activity. The polyproline type II helical bundle fold of the 9.6-kDa springtail (Collembola) AFP from Granisotoma rainieri (a primitive arthropod) facilitates changes to both IBS length and width. A one quarter decrease in area reduced activity to less than 10%. A one quarter increase in IBS width, through the addition of a single helix, tripled antifreeze activity. However, increasing IBS length by a similar amount actually reduced activity. Expanding the IBS area can greatly increase antifreeze activity but needs to be evaluated by experimentation on a case-by-case basis.

Revealing the Mechanism of Irreversible Binding of Antifreeze Glycoproteins to Ice.

Antifreeze glycoproteins (AFGPs) are a special kind of antifreeze proteins with strong flexibility. Whether their antifreeze activity is achieved by reversibly or irreversibly binding to ice is widely debated, and the molecular mechanism of irreversible binding remains unclear. In this work, the antifreeze mechanism of the smallest AFGP isoform, AFGP8, is investigated at the atomic level. The results indicate that AFGP8 can bind to ice both reversibly through its hydrophobic methyl groups (peptide binding) and irreversibly through its hydrophilic disaccharide moieties (saccharide binding). Although peptide binding occurs faster than saccharide binding, free-energy calculations indicate that the latter is energetically more favorable. In saccharide binding, at least one disaccharide moiety is frozen in the grown ice, resulting in irreversible binding, while the other moieties significantly perturb the water hydrogen-bonding network, thus inhibiting ice growth more effectively. The present study reveals the coexistence of reversible and irreversible bindings of AFGP8, both contributing to the inhibition of ice growth and further provides molecular mechanism of irreversible binding.

Submilligram Level of Beetle Antifreeze Proteins Minimize Cold-Induced Cell Swelling and Promote Cell Survival.

Hypothermic (cold) preservation is a limiting factor for successful cell and tissue transplantation where cell swelling (edema) usually develops, impairing cell function. University of Wisconsin (UW) solution, a standard cold preservation solution, contains effective components to suppress hypothermia-induced cell swelling. Antifreeze proteins (AFPs) found in many cold-adapted organisms can prevent cold injury of the organisms. Here, the effects of a beetle AFP from Dendroides canadensis (DAFP-1) on pancreatic ß-cells preservation were first investigated. As low as 500 µg/mL, DAFP-1 significantly minimized INS-1 cell swelling and subsequent cell death during 4 °C preservation in UW solution for up to three days. However, such significant cytoprotection was not observed by an AFP from Tenebrio molitor (TmAFP), a structural homologue to DAFP-1 but lacking arginine, at the same levels. The cytoprotective effect of DAFP-1 was further validated with the primary ß-cells in the isolated rat pancreatic islets in UW solution. The submilligram level supplement of DAFP-1 to UW solution significantly increased the islet mass recovery after three days of cold preservation followed by rewarming. The protective effects of DAFP-1 in UW solution were discussed at a molecular level. The results indicate the potential of DAFP-1 to enhance cell survival during extended cold preservation.

Identification of Novel Antifreeze Peptides from Takifugu obscurus Skin and Molecular Mechanism in Inhibiting Ice Crystal Growth.

Foodborne hydrolyzed antifreeze peptides have been widely used in the food industry and the biomedical field. However, the components of hydrolyzed peptides are complex and the molecular mechanism remains unclear. This study focused on identification and mechanism analysis of novel antifreeze peptides from Takifugu obscurus skin by traditional methods and computer-assisted techniques. Results showed that three peptides (EGPRAGGAPG, GDAGPSGPAGPTG, and GEAGPAGPAG) possessed cryoprotection via reducing the freezing point and inhibiting ice crystal growth. Molecular docking confirmed that the cryoprotective property was related to peptide structure, especially α-helix, and hydrogen bond sites. Moreover, the antifreeze peptides were double-faces, which controlled ice crystals while affecting the arrangement of surrounding water molecules, thus exhibiting a strong antifreeze activity. This investigation deepens the comprehension of the mechanism of antifreeze peptides at molecular scale, and the novel efficient antifreeze peptides can be developed in antifreeze materials design and applied in food industry.

Design and Analysis of a Mutant form of the Ice-Binding Protein from Choristoneura fumiferana.

Ice-binding proteins are expressed in the cells of some cold adapted organisms, helping them to survive at extremely low temperatures. One of the problems in studying such proteins is the difficulty of their isolation and purification. For example, eight cysteine residues in the cfAF (antifreeze protein from the eastern spruce budworm Choristoneura fumiferana) form intermolecular bridges during the overexpression of this protein. This impedes the process of the protein purification dramatically. To overcome this issue, in this work, we designed a mutant form of the ice-binding protein cfAFP, which is much easier to isolate that the wild-type protein. The mutant form named mIBP83 did not lose the ability to bind to ice surface. Besides, observation of the processes of freezing and melting of ice in the presence of mIBP83 showed that this protein affects the process of ice melting, increasing its melting temperature, and does not decrease the water freezing temperature.

Insight into polyproline II helical bundle stability in an antifreeze protein denatured state.

The use of polyproline II (PPII) helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. Here, circular dichroism spectroscopy revealed minor populations of PPII like conformers at low temperature. To get atomic level information on the conformational ensemble and entropy of the reduced, denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at four experimental conditions. No significant populations of stable secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH and shifted pKa values leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation the C1-C28 disulfide bond during nascent folding, although no evidence for preferred contacts between these positions was detected by paramagnetic relaxation enhancement under denaturing conditions. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured α/ß proteins both on fast ps/ns as well as slower µs/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.

Crystal waters on the nine polyproline type II helical bundle springtail antifreeze protein from Granisotoma rainieri match the ice lattice.

A springtail (Collembola) identified as Granisotoma rainieri was collected from snow in Hokkaido, Japan, in late winter when nighttime temperatures were below zero. Extracts of these arthropods showed antifreeze activity by shaping ice crystals and stopping their growth. The glycine-rich proteins responsible for this freezing point depression were isolated by ice-affinity purification and had principal masses of ~ 6.9 and 9.6 kDa. We identified a transcript for a 9.6-kDa component and produced it as a His-tagged recombinant protein for structural analysis. Its crystal structure was solved to a resolution of 1.21 Å and revealed a polyproline type II helical bundle, similar to the six-helix Hypogastrura harveyi AFP, but with nine helices organized into two layers held together by an extensive network of hydrogen bonds. One of the layers is flat, regular, and hydrophobic and likely serves as the ice-binding side. Although this surface makes close protein-protein contacts with its symmetry mate in the crystal, it has bound chains of waters present that resemble those on the basal and primary prism planes of ice. Molecular dynamic simulations indicate most of these crystal waters would preferentially occupy these sites if exposed to bulk solvent in the absence of the symmetry mate. These prepositioned waters lend further support to the ice-binding mechanism in which AFPs organize ice-like waters on one surface to adsorb to ice. DATABASES: Structural data are available in the Protein Data Bank under the accession number 7JJV. Transcript data are available in GenBank under accession numbers MT780727, MT780728, MT780729, MT780730, MT780731 and MT985982.

Poly(l-Ala-co-l-Lys) Exhibits Excellent Ice Recrystallization Inhibition Activity.

The control of ice recrystallization is very important in cryo-technological fields such as the food industry, biopharmaceuticals, and cell storage. Ice recrystallization inhibition (IRI) compounds are therefore designed to limit the growth of ice crystals, decrease the crystal size, and control the crystal shape. To improve the IRI activity of cryo-systems, various synthetic polymers such as biomimetic polypeptides from polar fish, facially amphiphilic polymers, polyampholytes, poly(vinyl alcohol) derivatives, and block copolymers with hydrophilic-hydrophobic balance have been developed. Except for graphene oxide, poly(vinyl alcohol) has thus far exhibited the best performance among these polymers. Herein, poly(l-alanine-co-l-lysine) (PAK) was shown to exhibit a similar IRI activity to that of poly(vinyl alcohol). Moreover, in contrast to the needle-shaped ice crystals generated by the aqueous PVA solution, the PAK solution was shown to generate cubic-to-spherical shaped ice crystals. Furthermore, neither poly(l-alanine-co-l-aspartic acid) (PAD) nor poly(ethylene glycol) (PEG) with a similar molecular weight provided any significant IRI activity. Examination by FTIR and circular dichroism spectroscopies indicated that the PAK forms α-helices, whereas the PAD forms random coils in water. Further, a dynamic ice shaping study suggested that PAK strongly interacts with ice crystals, whereas PAD and PEG only weakly interact. These results suggest that PAK is an important compound with superior IRI activity and that this activity is dependent upon the functional groups and secondary structure of the polypeptides.

Antifreeze Proteins and Their Practical Utilization in Industry, Medicine, and Agriculture.

Antifreeze proteins (AFPs) are specific proteins, glycopeptides, and peptides made by different organisms to allow cells to survive in sub-zero conditions. AFPs function by reducing the water's freezing point and avoiding ice crystals' growth in the frozen stage. Their capability in modifying ice growth leads to the stabilization of ice crystals within a given temperature range and the inhibition of ice recrystallization that decreases the drip loss during thawing. This review presents the potential applications of AFPs from different sources and types. AFPs can be found in diverse sources such as fish, yeast, plants, bacteria, and insects. Various sources reveal different α-helices and ß-sheets structures. Recently, analysis of AFPs has been conducted through bioinformatics tools to analyze their functions within proper time. AFPs can be used widely in various aspects of application and have significant industrial functions, encompassing the enhancement of foods' freezing and liquefying properties, protection of frost plants, enhancement of ice cream's texture, cryosurgery, and cryopreservation of cells and tissues. In conclusion, these applications and physical properties of AFPs can be further explored to meet other industrial players. Designing the peptide-based AFP can also be done to subsequently improve its function.

Demonstration of the cryoprotective properties of the fucose-containing polysaccharide FucoPol.

We report the cryoprotective potential of FucoPol, a fucose-containing bacterial exopolysaccharide produced by Enterobacter A47. In vitro cryopreservation assays of Vero, Saos-2, HFFF2 and C2C12 cell lines exposed to a validated non-cytotoxic 2.5 mg/mL FucoPol concentration demonstrated a consistent post-thaw metabolic viability increase. Calorimetric analysis showed a non-colligative, FucoPol concentration-dependent increase of the freezing point (Tf), with minimal change in melting point (Tm). Freezing point variation was corroborated by Polarized Optical Microscopy studies, also showing a reduction of ice crystal dimensions. Its proven shear-thinning behaviour and polyanionicity favour interactivity between the polysaccharide and the water-ice interface, resulting in ice growth inhibition. These findings demonstrate FucoPol's high promise as a bio-based, biodegradable approach to be implemented into cryopreservation formulations.

Bioinspired l-Proline Oligomers for the Cryopreservation of Oocytes via Controlling Ice Growth.

Various types of cells are routinely cryopreserved in modern regenerative and cell-based medicines. For instance, the oocyte is one of the most demanding cells to be cryopreserved in genetic engineering and human-assisted reproductive technology (ART). However, the usage of cryopreserved oocytes in ART clinics is still limited mainly because of the unstable survival rate. This is due to the fact that oocytes are more prone to be damaged by ice crystals in comparison to other cells, as oocytes are larger in size and surface area. Meanwhile, oocytes contain more water, and thus, ice crystals are easier to form inside the cells. Currently, to avoid injury by the formed ice crystals, cryopreservation (CP) of oocytes has to use large amounts of small molecules as cryoprotectants such as dimethyl sulfoxide (DMSO) and ethylene glycol (EG), which can permeate into the cell and prevent ice formation inside. However, these molecules are chemically and epigenetically toxic to cells. Therefore, great efforts have been focused on reducing the amount of DMSO and EG used for oocyte CP. In nature, the antifreeze (glyco)proteins (AFGPs) locate extracellularly with the ability to protect living organisms from freezing damage via controlling ice growth. Inspired by this, biocompatible and nontoxic L-proline oligomers (L-Pron), which have the same polyproline II helix structure as that of AFGPs, are first employed for the CP of oocytes. The experimental results reveal that L-Pro8 has a profound activity in inhibiting ice growth as that of AFGP8. Also, by the addition of 50 mM L-Pro8, the amount of DMSO and EG can be greatly reduced by ca. 1.8 M for oocyte CP; moreover, the survival rate of the cryopreserved oocytes is increased up to 99.11%, and the coefficient of variance of the survival rate is decreased from 7.47 to 2.15%. These results mean that almost all oocytes can survive after CP with our method; importantly, the mitochondrial function as a critical criterion for the quality of the frozen-thawed oocytes is also improved. It is proposed that with the addition of L-Pro8, the extracellular ice growth is slowed down, which prevents the direct injuries of cells by large ice crystals and the accompanying osmotic pressure increase. As such, this work is not only significant for meeting the ever-increasing demand by the ART clinics but also gives guidance for designing materials in controlling ice growth during CP of other cells and tissues.

The Inhibition of Icing and Frosting on Glass Surfaces by the Coating of Polyethylene Glycol and Polypeptide Mimicking Antifreeze Protein.

The development of anti-icing, anti-frosting transparent plates is important for many reasons, such as poor visibility through the ice-covered windshields of vehicles. We have fabricated new glass surfaces coated with polypeptides which mimic a part of winter flounder antifreeze protein. We adopted glutaraldehyde and polyethylene glycol as linkers between these polypeptides and silane coupling agents applied to the glass surfaces. We have measured the contact angle, the temperature of water droplets on the cooling surfaces, and the frost weight. In addition, we have conducted surface roughness observation and surface elemental analysis. It was found that peaks in the height profile, obtained with the atomic force microscope for the polypeptide-coated surface with polyethylene glycol, were much higher than those for the surface without the polypeptide. This shows the adhesion of many polypeptide aggregates to the polyethylene glycol locally. The average supercooling temperature of the droplet for the polypeptide-coated surface with the polyethylene glycol was lower than for the polypeptide-coated surface with glutaraldehyde and the polyethylene-glycol-coated surface without the polypeptide. In addition, the average weight of frost cover on the specimen was lowest for the polypeptide-coated surface with the polyethylene glycol. These results argue for the effects of combined polyethylene glycol and polypeptide aggregates on the locations of ice nuclei and condensation droplets. Thus, this polypeptide-coating with the polyethylene glycol is a potential contender to improve the anti-icing and anti-frosting of glasses.