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.

Peptidic Antifreeze Materials: Prospects and Challenges.

Necessitated by the subzero temperatures and seasonal exposure to ice, various organisms have developed a remarkably effective means to survive the harsh climate of their natural habitats. Their ice-binding (glyco)proteins keep the nucleation and growth of ice crystals in check by recognizing and binding to specific ice crystal faces, which arrests further ice growth and inhibits ice recrystallization (IRI). Inspired by the success of this adaptive strategy, various approaches have been proposed over the past decades to engineer materials that harness these cryoprotective features. In this review we discuss the prospects and challenges associated with these advances focusing in particular on peptidic antifreeze materials both identical and akin to natural ice-binding proteins (IBPs). We address the latest advances in their design, synthesis, characterization and application in preservation of biologics and foods. Particular attention is devoted to insights in structure-activity relations culminating in the synthesis of de novo peptide analogues. These are sequences that resemble but are not identical to naturally occurring IBPs. We also draw attention to impactful developments in solid-phase peptide synthesis and 'greener' synthesis routes, which may aid to overcome one of the major bottlenecks in the translation of this technology: unavailability of large quantities of low-cost antifreeze materials with excellent IRI activity at (sub)micromolar concentrations.

Expression of Ice-Binding Proteins in Caenorhabditis elegans Improves the Survival Rate upon Cold Shock and during Freezing.

Ice-binding proteins (IBPs) are capable of binding ice crystals and inhibiting their growth at freezing temperatures. IBPs are also thought to stabilize the cell membrane at non-freezing temperatures near 0 °C. These two effects have been assumed to reduce cold- and freezing-induced damage to cells and tissues. However, knowledge regarding the effects of IBP on the living animals is limited. Here, we characterized the relationship between the IBP effects and the physiological role by using the nematode Caenorhabditis elegans. The expression of fish (NfeIBPs)- and fungus-derived IBPs (AnpIBPs and TisIBP8) in C. elegans improved its survival rate during exposure to 0 and -2 °C (cold shock) and -5 °C (freezing). The observed cold tolerance of C. elegans after cold shock is attributable to the stabilization of cell-membrane lipids with IBPs, and the freezing tolerance at -5 °C can be attributed to the inhibition of ice-crystal growth by the IBPs. Significantly, the survival rate of C. elegans at -5 °C was improved by expression of wild-type AnpIBP and maximized by that of TisIBP8, whereas it was lowered when a defective AnpIBP mutant was expressed. These results suggest that the ice-binding ability of IBP has a good correlation with the survival rate of C. elegans during freezing.

Beetle, Dendroides canadensis, antifreeze proteins increased high temperature survivorship in transgenic fruit flies, Drosophila melanogaster.

Paradoxically, some insects have an increased capacity to survive higher temperatures in winter than summer. Possible contributors to this increased heat tolerance in winter could be their sub-zero adaptations (high polyol concentrations, antifreeze proteins, antifreeze glycolipids, etc.). To investigate if a sub-zero adaptation can increase organismal high temperature survivorship, we tested transgenic fruit flies, Drosophila melanogaster, with antifreeze proteins from the fire-colored beetle, Dendroides canadensis (DAFPs). Transgenic Drosophila melanogaster with individual DAFPs-1 and -4 had increased survivorship compared to control flies after 24 h when placed at 35-36.5 °C. The 24 h ULT50 (Upper Lethal Temperature at which 50% mortality occurred) was calculated to be 36.3 °C for DAFP-1 flies, 36.2 °C for DAFP-4 flies, 35.4 °C for wild-type controls, and 34.9 °C for GAL4 controls. The results indicate that DAFPs may have an alternative function in insects and be a contributor in the unexpected phenomenon of increased higher temperature survivorship in winter.

Protein evolution revisited.

Antifreeze proteins (AFPs) protect marine fishes from freezing in icy seawater. They evolved relatively recently, most likely in response to the formation of sea ice and Cenozoic glaciations that occurred less than 50 million years ago, following a greenhouse Earth event. Based on their diversity, AFPs have independently evolved on many occasions to serve the same function, with some remarkable examples of convergent evolution at the structural level, and even instances of lateral gene transfer. For some AFPs, the progenitor gene is recognizable. The intense selection pressure exerted by icy seawater, which can rapidly kill unprotected fish, has led to massive AFP gene amplification, as well as some partial gene duplications that have increased the size and activity of the antifreeze. The many protein evolutionary processes described in Gordon H. Dixon's Essays in Biochemistry article will be illustrated here by examples from studies on AFPs. Abbreviations: AFGP: antifreeze glycoproteins; AFP: antifreeze proteins; GHD: Gordon H. Dixon; SAS: sialic acid synthase; TH: thermal hysteresis.

Mechanisms underlying insect freeze tolerance.

Freeze tolerance - the ability to survive internal ice formation - has evolved repeatedly in insects, facilitating survival in environments with low temperatures and/or high risk of freezing. Surviving internal ice formation poses several challenges because freezing can cause cellular dehydration and mechanical damage, and restricts the opportunity to metabolise and respond to environmental challenges. While freeze-tolerant insects accumulate many potentially protective molecules, there is no apparent 'magic bullet' - a molecule or class of molecules that appears to be necessary or sufficient to support this cold-tolerance strategy. In addition, the mechanisms underlying freeze tolerance have been minimally explored. Herein, we frame freeze tolerance as the ability to survive a process: freeze-tolerant insects must withstand the challenges associated with cooling (low temperatures), freezing (internal ice formation), and thawing. To do so, we hypothesise that freeze-tolerant insects control the quality and quantity of ice, prevent or repair damage to cells and macromolecules, manage biochemical processes while frozen/thawing, and restore physiological processes post-thaw. Many of the molecules that can facilitate freeze tolerance are also accumulated by other cold- and desiccation-tolerant insects. We suggest that, when freezing offered a physiological advantage, freeze tolerance evolved in insects that were already adapted to low temperatures or desiccation, or in insects that could withstand small amounts of internal ice formation. Although freeze tolerance is a complex cold-tolerance strategy that has evolved multiple times, we suggest that a process-focused approach (in combination with appropriate techniques and model organisms) will facilitate hypothesis-driven research to understand better how insects survive internal ice formation.

Identification and Characterization of an Isoform Antifreeze Protein from the Antarctic Marine Diatom, Chaetoceros neogracile and Suggestion of the Core Region.

Antifreeze proteins (AFPs) protecting the cells against freezing are produced in response to extremely low temperatures in diverse psychrophilic organisms, and they are encoded by multiple gene families. The AFP of Antarctic marine diatom Chaetoceros neogracile is reported in our previous research, but like other microalgae, was considered to probably have additional genes coding AFPs. In this paper, we reported the cloning and characterization of additional AFP gene from C. neogracile (Cn-isoAFP). Cn-isoAFP protein is 74.6% identical to the previously reported Cn-AFP. The promoter sequence of Cn-isoAFP contains environmental stress responsive elements for cold, thermal, and high light conditions. Cn-isoAFP transcription levels increased dramatically when cells were exposed to freezing (-20 °C), thermal (10 °C), or high light (600 µmol photon m-2 s-1) stresses. The thermal hysteresis (TH) activity of recombinant Cn-isoAFP was 0.8 °C at a protein concentration of 5 mg/mL. Results from homology modeling and TH activity analysis of site-directed mutant proteins elucidated AFP mechanism to be a result of flatness of B-face maintained via hydrophobic interactions.

Functional Innovations and the Conquest of the Oceans by Acanthomorph Fishes.

The world's oceans are home to many fantastic creatures, including about 16,000 species of actinopterygian, or ray-finned, fishes. Notably, 85% of marine fish species come from a single actinopterygian subgroup, the acanthomorph or spiny-rayed fishes. Here, we review eight functional innovations found in marine acanthomorphs that have been instrumental in the adaptive radiation of this group in the marine realm. Jaw protrusion substantially enhances the suction feeding mechanism found in all fish. Fin spines serve as a major deterrent to predators and enhance the locomotor function of fins. Pharyngognathy, a specialization of the second pair of jaws in the pharynx, enhances the ability of fishes to process hard and tough prey. Endothermy allows fishes to function at high levels of physiological performance in cold waters and facilitates frequent movement across strong thermal gradients found in the open ocean. Intramandibular joints enhance feeding for fishes that bite and scrape prey attached to hard surfaces. Antifreeze proteins prevent ice crystal growth in extracellular fluids, allowing fish to function in cold waters that would otherwise freeze them. Air-breathing allowed fishes at the water's edge to exploit terrestrial habitats. Finally, bioluminescence functions in communication, attracting prey and in hiding from predators, particularly for fishes of the deep ocean. All of these innovations have evolved multiple times in fishes. The frequent occurrence of convergent evolution of these complex functional novelties speaks to the persistence and potency of the selective forces in marine environments that challenge fishes and stimulate innovation.

Neofunctionalization of zona pellucida proteins enhances freeze-prevention in the eggs of Antarctic notothenioids.

The mechanisms by which the eggs of the Antarctic notothenioid fishes avoid freezing are not fully understood. Zona pellucida proteins (ZPs) are constituents of the chorion which forms a protective matrix surrounding the egg. Here we report occurrence of freezing temperature-related gene expansion and acquisition of unusual ice melting-promoting (IMP) activity in a family of Antarctic notothenioid ZPs (AnnotoZPs). Members of AnnotoZPs are shown to bind with ice and non-colligatively depress the melting point of a solution in a range of 0.26 to 0.65 °C at a moderate concentration. Eggs of zebrafishes expressing an AnnotoZP transgene show improved melting point depression and enhanced survival in freezing conditions. Mutational analyses in a representative AnnotoZP indicate the ZP domain and patches of acidic residues are essential structures for the IMP activity. AnnotoZPs, therefore, represent a group of macromolecules that prevent freezing by a unique ZP-ice interaction mechanism distinct from the known antifreeze proteins.

Antarctic notothenioid fish: what are the future consequences of 'losses' and 'gains' acquired during long-term evolution at cold and stable temperatures?

Antarctic notothenioids dominate the fish fauna of the Southern Ocean. Evolution for millions of years at cold and stable temperatures has led to the acquisition of numerous biochemical traits that allow these fishes to thrive in sub-zero waters. The gain of antifreeze glycoproteins has afforded notothenioids the ability to avert freezing and survive at temperatures often hovering near the freezing point of seawater. Additionally, possession of cold-adapted proteins and membranes permits them to sustain appropriate metabolic rates at exceptionally low body temperatures. The notothenioid genome is also distinguished by the disappearance of traits in some species, losses that might prove costly in a warmer environment. Perhaps the best-illustrated example is the lack of expression of hemoglobin in white-blooded icefishes from the family Channichthyidae. Loss of key elements of the cellular stress response, notably the heat shock response, has also been observed. Along with their attainment of cold tolerance, notothenioids have developed an extreme stenothermy and many species perish at temperatures only a few degrees above their habitat temperatures. Thus, in light of today's rapidly changing climate, it is critical to evaluate how these extreme stenotherms will respond to rising ocean temperatures. It is conceivable that the remarkable cold specialization of notothenioids may ultimately leave them vulnerable to future thermal increases and threaten their fitness and survival. Within this context, our review provides a current summary of the biochemical losses and gains that are known for notothenioids and examines these cold-adapted traits with a focus on processes underlying thermal tolerance and acclimation capacity.

Antifreeze proteins enable plants to survive in freezing conditions.

Overwintering plants secrete antifreeze proteins (AFPs) to provide freezing tolerance. These proteins bind to and inhibit the growth of ice crystals that are formed in the apoplast during subzero temperatures. Antifreeze activity has been detected in more than 60 plants and AFPs have been purified from 15 of these, including gymnosperms, dicots and monocots. Biochemical characterization of plant antifreeze activity, as determined by the high ice recrystallization inhibition (IRI) activities and low thermal hysteresis (TH) of AFPs, showed that their main function is inhibition of ice crystal growth rather than the lowering of freezing temperatures. However, recent studies showed that antifreeze activity with higher TH also exists in plants. Calcium and hormones like ethylene and jasmonic acid have been shown to regulate plant antifreeze activity. Recent studies have shown that plant AFPs bind to both prism planes and basal planes of ice crystals by means of two flat ice binding sites. Plant AFPs have been postulated to evolve from the OsLRR-PSR gene nearly 36 million years ago. In this review, we present the current scenario of plant AFP research in order to understand the possible potential of plant AFPs in generation of freezing-tolerant crops.

The role of sulfates on antifreeze protein activity.

In the present study, we have investigated the effect of sodium sulfate (Na2SO4) buffer on the antifreeze activity of DAFP-1, the primary AFP in the hemolymph of the beetle Dendroides canadensis. In contrast to previous studies, we found evidence that sodium sulfate does not suppress antifreeze activity of DAFP-1. Terahertz absorption spectroscopy (THz) studies were combined with molecular dynamics (MD) simulations to investigate the change in collective hydrogen bond dynamics in the vicinity of the AFP upon addition of sodium sulfate. The MD simulations revealed that the gradient of H-bond dynamics toward the ice-binding site is even more pronounced when adding sodium sulfate: The cosolute dramatically slows the hydrogen bond dynamics on the ice-binding plane of DAFP-1, whereas it has a more modest effect in the vicinity of other parts of the protein. These theoretical predictions are in agreement with the experimentally observed increase in THz absorption for solvated DAFP-1 upon addition of sodium sulfate. These studies support our previously postulated mechanism for AF activity, with a preferred ice binding by threonine on nanoice crystals which is supported by a long-range effect on hydrogen bond dynamics.

Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca(2+) levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+)-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.

New insights into ice growth and melting modifications by antifreeze proteins.

Antifreeze proteins (AFPs) evolved in many organisms, allowing them to survive in cold climates by controlling ice crystal growth. The specific interactions of AFPs with ice determine their potential applications in agriculture, food preservation and medicine. AFPs control the shapes of ice crystals in a manner characteristic of the particular AFP type. Moderately active AFPs cause the formation of elongated bipyramidal crystals, often with seemingly defined facets, while hyperactive AFPs produce more varied crystal shapes. These different morphologies are generally considered to be growth shapes. In a series of bright light and fluorescent microscopy observations of ice crystals in solutions containing different AFPs, we show that crystal shaping also occurs during melting. In particular, the characteristic ice shapes observed in solutions of most hyperactive AFPs are formed during melting. We relate these findings to the affinities of the hyperactive AFPs for the basal plane of ice. Our results demonstrate the relation between basal plane affinity and hyperactivity and show a clear difference in the ice-shaping mechanisms of most moderate and hyperactive AFPs. This study provides key aspects associated with the identification of hyperactive AFPs.

Ancient climate change, antifreeze, and the evolutionary diversification of Antarctic fishes.

The Southern Ocean around Antarctica is among the most rapidly warming regions on Earth, but has experienced episodic climate change during the past 40 million years. It remains unclear how ancient periods of climate change have shaped Antarctic biodiversity. The origin of antifreeze glycoproteins (AFGPs) in Antarctic notothenioid fishes has become a classic example of how the evolution of a key innovation in response to climate change can drive adaptive radiation. By using a time-calibrated molecular phylogeny of notothenioids and reconstructed paleoclimate, we demonstrate that the origin of AFGP occurred between 42 and 22 Ma, which includes a period of global cooling approximately 35 Ma. However, the most species-rich lineages diversified and evolved significant ecological differences at least 10 million years after the origin of AFGPs, during a second cooling event in the Late Miocene (11.6-5.3 Ma). This pattern indicates that AFGP was not the sole trigger of the notothenioid adaptive radiation. Instead, the bulk of the species richness and ecological diversity originated during the Late Miocene and into the Early Pliocene, a time coincident with the origin of polar conditions and increased ice activity in the Southern Ocean. Our results challenge the current understanding of the evolution of Antarctic notothenioids suggesting that the ecological opportunity that underlies this adaptive radiation is not linked to a single trait, but rather to a combination of freeze avoidance offered by AFGPs and subsequent exploitation of new habitats and open niches created by increased glacial and ice sheet activity.

How will fish that evolved at constant sub-zero temperatures cope with global warming? Notothenioids as a case study.

Current climate change has raised concerns over the fate of the stenothermal Antarctic marine fauna (animals that evolved to live in narrow ranges of cold temperatures). The present paper focuses on Notothenioidei, a taxonomic group that dominates Antarctic fish. Notothenioids evolved in the Southern Ocean over the last 20 million years, providing an example of a marine species flock with unique adaptations to the cold at morphological, physiological and biochemical levels. Their phenotypic modifications are often accompanied by 'irreversible' genomic losses or gene amplifications. On a micro-evolutionary scale, relatively 'shallow' genetic variation is observed, on account of past fluctuations in population size, and a significant genetic structure is evident, suggesting low population connectivity. These features suggest that Antarctic fish might have relatively little potential to adapt to global warming, at least at a genetic level. The extent of their phenotypic plasticity, which is evident to some degree, awaits further research.

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.

Arginine, a key residue for the enhancing ability of an antifreeze protein of the beetle Dendroides canadensis.

Antifreeze proteins (AFPs) can produce a difference between the nonequilibrium freezing point and the melting point, termed thermal hysteresis (TH). The TH activity of an antifreeze protein (AFP) depends on the specific AFP and its concentration as well as the presence of cosolutes including low molecular mass solutes and/or proteins. We recently identified series of carboxylates and polyols as efficient enhancers for an AFP from the beetle Dendroides canadensis. In this study, we chemically modified DAFP-1 using the arginine-specific reagent 1,2-cyclohexanedione. We demonstrated that 1,2-cyclohexanedione specifically modifies one arginine residue and the modified DAFP-1 loses its enhancing ability completely or partially in the presence of previously identified enhancers. The stronger the enhancement ability of the enhancer on the native DAFP-1, the stronger the enhancement effect of the enhancer on the modified DAFP-1. The weaker enhancers (e.g., glycerol) completely lose their enhancement effect on the modified DAFP-1 due to their inability to compete with 1,2-cyclohexanedione for the arginine residue. Regeneration of the arginine residue using hydroxylamine fully restored the enhancing ability of DAFP-1. These studies indicated that an arginine residue is critical for the enhancing ability of DAFP-1 and the guanidinium group of the arginine residue is important for its interaction with the enhancers, where the general mechanism of arginine-ligand interaction is borne. This work may initiate a complete mechanistic study of the enhancement effect in AFPs.

Ice recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic Antarctic hair grass Deschampsia antarctica E. Desv.

Antarctic hair grass (Deschampsia antarctica E. Desv.), the only grass indigenous to Antarctica, has well-developed freezing tolerance, strongly induced by cold acclimation. Here, we show that in response to low temperatures, D. antarctica expresses potent recrystallization inhibition (RI) activity that, inhibits the growth of small ice crystals into potentially damaging large ones, is proteinaceous and localized to the apoplasm. A gene family from D. antarctica encoding putative homologs of an ice recrystallization inhibition protein (IRIP) has been isolated and characterized. IRIPs are apoplastically targeted proteins with two potential ice-binding motifs: 1-9 leucine-rich repeats (LRRs) and c. 16 'IRIP' repeats. IRIP genes appear to be confined to the grass subfamily Pooideae and their products, exhibit sequence similarity to phytosulphokine receptors and are predicted to adopt conformations with two ice-binding surfaces. D. antarctica IRIP (DaIRIP) transcript levels are greatly enhanced in leaf tissue following cold acclimation. Transgenic Arabidopsis thaliana expressing a DaIRIP has novel RI activity, and purified DaIRIP, when added back to extracts of leaves from non-acclimated D. antarctica, can reconstitute the activity found in acclimated plants. We propose that IRIP-mediated RI activity may contribute to the cryotolerance of D. antarctica, and thus to its unique ability to have colonized Antarctica.

Salt-induced enhancement of antifreeze protein activity: a salting-out effect.

Antifreeze proteins are a structurally diverse group of proteins characterized by their unique ability to cause a separation of the melting- and growth-temperatures of ice. These proteins have evolved independently in different kinds of cold-adapted ectothermic animals, including insects and fish, where they protect against lethal freezing of the body fluids. There is a great variability in the capacity of different kinds of antifreeze proteins to evoke the antifreeze effect, but the basis of these differences is not well understood. This study reports on salt-induced enhancement of the antifreeze activity of an antifreeze protein from the longhorn beetle Rhagium inquisitor (L.). The results imply that antifreeze activity is predetermined by a steady-state distribution of the antifreeze protein between the solution and the ice surface region. The observed salt-induced enhancement of the antifreeze activity compares qualitatively and quantitatively with salt-induced lowering of protein solubility. Thus, salts apparently enhance antifreeze activity by evoking a solubility-induced shift in the distribution pattern of the antifreeze proteins in favour of the ice. These results indicate that the solubility of antifreeze proteins in the solution surrounding the ice crystal is a fundamental physiochemical property in relation to their antifreeze potency.