A comprehensive evaluation of the effects and mechanisms of antifreeze proteins during low-temperature preservation

During the past 10 years, it has become clear that the effects of antifreeze proteins (AFPs) on cell viability and on thermodynamic properties during low-temperature preservation are complex, even controversial. In this paper, these studies are reviewed systematically and some conclusions are drawn. It is shown that AFPs can display both protective and cytotoxic actions and both nucleation of ice and inhibition of ice crystal growth, depending on several factors; these include the specific storage protocol, the dose and type of AFP, the composition and concentration of cryoprotectant, and the features of the biological material. A novel model, incorporating some recent findings concerning these proteins, is proposed to explain this dual effect of AFPs during cryopreservation. AFP-ice complexes have some affinity interactions with cell membranes and with many other molecules present in cryopreservation solutions. When the intensity of these interactions reaches a certain level, the AFP-ice complexes may be induced to aggregate, thereby inducing ice nucleation and loss of the ability to inhibit recrystallization.     

Ice Recrystallization Behavior of Corn Starch/Sucrose Solutions: Effects of Addition of Corn Starch and Antifreeze Protein III

Knowledge of the behavior of corn starch during frozen storage is necessary to understand more complex systems. In the present study, ice recrystallization in corn starch (0.3% and 3%, w/w)/sucrose (40%, w/w) solution was investigated at −10 °C based on the theory of Ostwald ripening. The addition of corn starch to the sucrose solution increased the ice recrystallization (IR) rate constant. To explore the mechanism causing higher IR rate constant, fluorescence microscopy was used to analyze the distribution of corn starch molecules. Fluorescence micrograph showed corn starch distributed homogenously in the freeze-concentrated phase. Ice crystal size distribution assessment showed that at the same average radius, the addition of corn starch increased the standard deviation of ice crystal size distribution. The findings revealed that the addition of corn starch widened the distribution of ice crystal size, which may be the mechanism causing higher IR rate constant. To inhibit the ice recrystallization process, antifreeze protein type III (AFP III) was added to sucrose solutions with and without corn starch. In the presence of corn starch, 0.01-mg/mL AFP III was enough to significantly reduce the IR rate. Conversely, the samples without corn starch did not show a significant reduction in IR rate constant at the same AFP III concentration. The outcomes revealed that corn starch enhanced the activity of AFP III. The results of this study showed that corn starch increased the IR rate constant, and AFP III supplemented with corn starch was synergistically more efficient in retarding IR rate constant.

     

Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants

Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly expressed in Escherichia coli strain BL21 (DE3). The activity of the purified and refolded recombinant proteins was analyzed by measurement of thermal hysteresis (TH) activity and detection of in vitro antifreeze activity by measuring enhanced cold resistance of bacteria. Two carrot AFP mutants generated by site-directed mutagenesis were also expressed and purified under these conditions for use in parallel experiments. Recombinant DcAFP displayed a TH activity equivalent to that of native DcAFP, while mutants DcAFP-N130Q and rDcAFP-N130V showed 32 and 43% decreases in TH activity, respectively. Both the recombinant DcAFP and its mutants were able to enhance the cold resistance of bacteria, to degrees consistent with their respective TH activities.     

抗冻蛋白应用于水稻悬浮细胞超低温保存的研究

AFP from winter flounder was utilized in cryopreservation of plant cells. During cryopreservation of rice suspension cells by two-step method, AFP at 0.01 mg/ml damaged the cells extremely. The data obtained at relatively high concentration, however, decreased the variability of survival rate. During vitrification of rice cells, AFP at 0.2 mg/ml enhanced the viability. However, high concentration AFP (> 5 mg/ml) decreased the recovery rate. Studies indicated that the results of application of AFP in cryopreservation were closely related to the concentration of cryoprotectant. The amount of ice crystal in environment, the concentration of AFP and cryoprotectant, and the composition of plasma membrane were several key factors affecting the results of AFP application. In mechanism analysis, the authors suggested that on one hand AFP can interact with ice crystal, which inhibits ice recrystallization and prevent the cells from devitrification. On the other hand, AFP also can interact with cell membrane, resulting in the ice growth around the plasma membrane.     

Effects of warming rate,temperature, and antifreeze proteins on the survival of mouse spermatozoa frozen at an optimal rate

We have recently reported that the survival of mouse spermatozoa is decreased when they are warmed at a suboptimal rate after being frozen at an optimal rate. We proposed that this drop in survival is caused by physical damage derived from the recrystallization of extracellular ice during slow warming. The first purpose of the present study was to determine the temperatures over which the decline in survival occurs during slow warming and the kinetics of the decline at fixed subzero temperatures. The second purpose was to examine the effects of antifreeze proteins (AFP) on the survival of slowly warmed mouse spermatozoa, the rationale being that AFP have the property of inhibiting ice recrystallization. With respect to the first point, a substantial loss in motility occurred when slow warming was continued to higher than -50 degrees C and the survival of the sperm decreased with an increase in the temperature at which slow warming was terminated. In contrast, the motility of sperm that were warmed rapidly to these temperatures remained high initially but dropped with increased holding time. At -30 degrees C, most of the drop occurred in 5 min. These results are consistent with the hypothesis that damage develops as a consequence of the recrystallization of the external ice. AFP ought to inhibit such recrystallization, but we found that the addition of AFP-I, AFP-III, and an antifreeze glycoprotein at concentrations of 1-100 microg/ml did not protect the frozen-thawed cells; rather it led to a decrease in survival that was proportional to the concentration. There was no decrease in survival from exposure to the AFP in the absence of freezing. AFP are known to produce changes in the structure and habit of ice crystals, and some have reported deleterious consequences associated with those structural changes. We suggest that such changes may be the basis of the adverse effects of AFP on the survival of the sperm, especially since mouse sperm are exquisitely sensitive to a variety of mechanical stresses.     

A leucine-rich repeat protein of carrot that exhibits antifreeze activity

A gene encoding an antifreeze protein (AFP) was isolated from carrot (Daucus carota) using sequence information derived from the purified protein. The carrot AFP is highly similar to the polygalacturonase inhibitor protein (PGIP) family of apoplastic plant leucine-rich repeat (LRR) proteins. Expression of the AFP gene is rapidly induced by low temperatures. Furthermore, expression of the AFP gene in transgenic Arabidopsis thaliana plants leads to an accumulation of antifreeze activity. Our findings suggest that a new type of plant antifreeze protein has recently evolved from PGIPs.     

Processing of ovine cardiac valve allografts: 1. Effects of preservation method on structure and mechanical properties.

It is essential to have some method of preservation of allograft valves during the time between procurement and implantation. Cryopreservation is the most commonly-used storage method today but it has the major disadvantage of high cost, and because its aim is to preserve living cells only relatively gentle antimicrobial treatments are used. This study addresses two interrelated questions: Is it necessary to maintain living donor cells in the tissue graft?Can more effective measures be used to reduce the risk of transmission of diseases, especially viral diseases, via human tissue grafts. In this paper, were port an investigation of four preservation methods that could be combined with more effective disinfection: cryopreservation with dimethyl sulphoxide, storage at ～4 °C in a high concentration of glycerol as used for the preservation of skin, snap-freezing by immersion in liquid nitrogen and vitrification. Snap freezing was mechanically damaging and vitrification proved to be impracticable but two methods, cryopreservation and storage in 85%glycerol, were judged worthy of further study. Cryopreservation was shown to maintain cellular viability and excellent microscopic structure with unchangedmechanical properties. The glycerol-preserved valves did not contain any living cells but the connective tissue matrix and mechanical properties were well preserved. The importance of living cells in allograft valves is uncertain. If living cells are unimportant then either method could be combined with more effective disinfection methods: in that case the simplicity and economy of the glycerol method would be advantageous. These questions are addressed in the two later papers in this series.     

Inhibiting ice recrystallization and optimization of cell viability after cryopreservation

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220?mM solutions of disaccharides; however, the best cell viability was obtained when a 200?mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.     

Effect of Long-Term Storage on Water Status and Physicochemical Properties of Nutritionally Enhanced Tortillas

The effect of functional ingredients (carrot juice, whole soy flour, and whole kamut flour) and storage (180 days) on physicochemical properties (texture and amylopectin recrystallization) and water status (moisture content, water activity, ice melting peak thermal properties, and proton nuclear magnetic resonance (¹H NMR) mobility) of tortillas has been studied. Different formulations significantly changed the parameters studied during storage resulting in larger changes than in the standard formulation (STD) that, therefore, may be considered the most stable product. The properties of whole kamut tortillas were very similar to those of standard sample while the formulation that contained carrot juice lead to an increased system rigidity observable both at macroscopic (textural properties), macromolecular (significantly reduced), and molecular (¹H FID) levels. A decrease of moisture content, water activity, endothermic transition ~0 °C, and an increase of ¹H NMR mobility (¹H T₂ pop A and C) were observed in soy-containing products [(soy enriched (SOY) and carrot, soy, and kamut (CSK)]. SOY and CSK had very low water activity, presented the highest ¹H NMR molecular mobility and underwent the most marked changes during storage suggesting that water activity cannot be taken as a sole indicator of food stability as very important modifications occurred in tortillas at molecular level.     

A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer

Cryopreservation of embryogenic tissue is an essential storage step in genotype selection and seedling production through somatic embryogenesis. To date, immature conifer somatic embryos, at the proliferation step, were only able to tolerate ultra low temperature after prior cryoprotectant treatments. We report a novel cryopreservation method for conifer (interior spruce and Douglas-fir) embryogenic tissue focusing on the maturation step of developing embryos that forgoes such cryoprotectant treatment. In this study, somatic embryos matured on culture media containing abscisic acid (ABA) at 20°C for 8 weeks. Typically, matured embryos in this manner were able to survive cryopreservation. The embryogenicity, however, decreased with increasing embryo maturity. Non-freezing low temperatures, such as 5°C, not only inhibited cotyledon development but also maintained embryogenicity. Cryotolerance was successfully induced when embryos were matured (or pretreated) under 5°C for a suitable culture period, typically 4–8 weeks. These embryos were able to survive a rapid cooling process and liquid nitrogen storage without the addition of any cryoprotectants. After cryopreservation, embryogenic tissue was recovered in both interior spruce and Douglas-fir. Embryo maturation tests indicated no difference in mature embryo yields with or without cryopreservation in interior spruce. The key factors inducing cryotolerance included ABA supplementation in culture media and low temperature pretreatment. Optimum combinations of these factors can result in high rates of tissue survival and high embryogenicity after cryopreservation.     

A theoretical model of a plant antifreeze protein from Lolium perenne.

Antifreeze proteins (AFPs), found in certain organisms enduring freezing environments, have the ability to inhibit damaging ice crystal growth. Recently, the repetitive primary sequence of the AFP of perennial ryegrass, Lolium perenne, was reported. This macromolecular antifreeze has high ice recrystallization inhibition activity but relatively low thermal hysteresis activity. We present here a theoretical three-dimensional model of this 118-residue plant protein based on a beta-roll domain with eight loops of 14-15 amino acids. The fold is supported by a conserved valine hydrophobic core and internal asparagine ladders at either end of the roll. Our model, which is the first proposed for a plant AFP, displays two putative, opposite-facing, ice-binding sites with surface complementarity to the prism face of ice. The juxtaposition of the two imperfect ice-binding surfaces suggests an explanation for the protein's inferior thermal hysteresis but superior ice recrystallization inhibition activity and activity when compared with fish and insect AFPs.     

Calorimetric determination of inhibition of ice crystal growth by antifreeze protein in hydroxyethyl starch solutions.

Differential scanning calorimetry and cryomicroscopy were used to investigate the effects of type I antifreeze protein (AFP) from winter flounder on 58% solutions of hydroxyethyl starch. The glass, devitrification, and melt transitions noted during rewarming were unaffected by 100 micrograms/ml AFP. Isothermal annealing experiments were undertaken to detect the effects of AFP-induced inhibition of ice crystal growth using calorimetry. A premelt endothermic peak was detected during warming after the annealing procedure. Increasing the duration or the temperature of the annealing for the temperature range from -28 and -18 degrees C resulted in a gradual increase in the enthalpy of the premelt endotherm. This transition was unaffected by 100 micrograms/ml AFP. Annealing between -18 and -10 degrees C resulted in a gradual decrease in the premelt peak enthalpy. This process was inhibited by 100 micrograms/ml AFP. Cryomicroscopic examination of the samples revealed that AFP inhibited ice recrystallization during isothermal annealing at -10 degrees C. Annealing at lower temperatures resulted in minimal ice recrystallization and no visible effect of AFP. Thus, the 100 micrograms/ml AFP to have a detectable influence on thermal events in the calorimeter, conditions must be used that result in significant ice growth without AFP and visible inhibition of this process by AFP.     

Antifreeze proteins and their potential use in frozen foods

Antifreeze proteins (AFPs) are proteins that have the ability to modify the growth of ice, resulting in the stabilization of ice crystals over a defined temperature range and in the inhibition of the recrystallization of ice. AFPs are found in a wide range of organisms, including bacteria, fungi, plants, invertebrates and fish. Moreover, multiple forms of AFPs are synthesized within each organism. As a result, it should be possible to select an AFP with appropriate characteristics and a suitable level of activity for a particular food product. Antifreeze proteins may improve the quality of foods that are eaten while frozen by inhibiting recrystallization and maintaining a smooth texture. In foods that are frozen only for preservation, AFPs may inhibit recrystallization during freezing, storage, transport and thawing, thus preserving food texture by reducing cellular damage and also minimizing the loss of nutrients by reducing drip. Antifreeze proteins are naturally present in many foods consumed as part of the human diet. However, AFPs may be introduced into other food products either by physical processes, such as mixing and soaking, or by gene transfer.     

Preservation of Pseudomonas putida bacteria at low temperatures]

We have found that the mode of cooling, composition of cryopreservation medium, original concentration of cells and storage temperature affect viability of Pseudomonas putida bacteria during low-temperature preservation. We have elaborated the conditions of preservation, providing for a high survival of bacteria, namely: one-stage cooling with the rates of 30, 40 degrees C/min or immersion into liquid nitrogen in the culturing medium with addition of sucrose, glycerol or dimexide in the concentration of 0.5 M; storage temperature is -80 degrees C divided by -196 degrees C.     

A facile method for determining ice recrystallization inhibition by antifreeze proteins

Ice recrystallization, the growth of large ice crystals at the expense of small ones, stresses freeze tolerant organisms and causes spoilage of frozen foods. This process is inhibited by antifreeze proteins (AFPs). Here, we present a simple method for determining the ice recrystallization inhibition (RI) activity of an AFP under physiological conditions using 10microl glass capillaries. Serial dilutions were prepared to determine the concentration below which RI activity was no longer detected, termed the RI endpoint. For type III AFP this was 200nM. The capillary method allows samples to be aligned and viewed simultaneously, which facilitates RI endpoint determination. Once prepared, the samples can be used reproducibly in subsequent RI assays and can be archived in a freezer for future reference. This method was used to detect the elution of type III AFP from a Sephadex G-75 size-exclusion column. RI activity was found at the expected V(e) for a 7kDa protein and also unexpectedly in the void volume.     

Expression of a cystine-rich fish antifreeze in transgenicDrosophila melanogaster

We have usedDrosophila melanogaster as a model system for the transgenic expression of cystine-rich Type II antifreeze protein (AFP) from sea raven. This protein was synthesized and secreted into fly haemolymph where it migrated as a larger species (16 kDa) than the mature form of the protein (14 kDa) as judged by immunoblotting.Drosophila-produced Type II AFP demonstrated antifreeze activity both in terms of thermal hysteresis (0.13 °C) and inhibition of ice recrystallization. Recombinant AFP was purified and N-terminal sequencing revealed a 17 aa extension that began at the predicted signal peptide cleavage point. The expression of all three AFP types in transgenicDrosophila has now been achieved. We conclude that the globular Type II and Type III AFPs are better choices for antifreeze transfer to other organisms than is the more widely used linear Type I AFP.     

Peptide backbone circularization enhances antifreeze protein thermostability

Antifreeze proteins (AFPs) are a class of ice‐binding proteins that promote survival of a variety of cold‐adapted organisms by decreasing the freezing temperature of bodily fluids. A growing number of biomedical, agricultural, and commercial products, such as organs, foods, and industrial fluids, have benefited from the ability of AFPs to control ice crystal growth and prevent ice recrystallization at subzero temperatures. One limitation of AFP use in these latter contexts is their tendency to denature and irreversibly lose activity at the elevated temperatures of certain industrial processing or large‐scale AFP production. Using the small, thermolabile type III AFP as a model system, we demonstrate that AFP thermostability is dramatically enhanced via split intein‐mediated N‐ and C‐terminal end ligation. To engineer this circular protein, computational modeling and molecular dynamics simulations were applied to identify an extein sequence that would fill the 20‐Å gap separating the free ends of the AFP, yet impose little impact on the structure and entropic properties of its ice‐binding surface. The top candidate was then expressed in bacteria, and the circularized protein was isolated from the intein domains by ice‐affinity purification. This circularized AFP induced bipyramidal ice crystals during ice growth in the hysteresis gap and retained 40% of this activity even after incubation at 100°C for 30 min. NMR analysis implicated enhanced thermostability or refolding capacity of this protein compared to the noncyclized wild‐type AFP. These studies support protein backbone circularization as a means to expand the thermostability and practical applications of AFPs.     

C-linked antifreeze glycoprotein (C-AFGP) analogues as novel cryoprotectants

Significant cell damage occurs during cryopreservation resulting in a decreased number of viable and functional cells post-thawing. Recent studies have correlated the unsuccessful outcome of regenerative therapies with poor cell viability after cryopreservation. Cell damage from ice recrystallization during freeze-thawing is one cause of decreased viability after cryopreservation. We have assessed the ability of two C-AFGPs that are potent inhibitors of ice recrystallization to increase cell viability after cryopreservation. Our results indicate that a 1-1.5 mg/mL (0.5-0.8 mM) solution of C-AFGP 1 is an excellent alternative to a 2.5% DMSO solution for the cryopreservation of human embryonic liver cells.