A Comprehensive Evaluation of the Effects and Mechanisms of Antifreeze Proteins during Low-Temperature Preservation

Cryobiology 41, 1–9 (2000) doi:10.1006/cryo.2000.2265, available online at http://www.idealibrary.com on

REVIEW ARTICLE A Comprehensive Evaluation of the Effects and Mechanisms of Antifreeze Proteins during Low-Temperature Preservation Jun-Hui Wang College of Life Science, Zhejiang University, Wensan Road, Hangzhou 310012, China 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. © 2000 Academic Press Key Words: antifreeze proteins; cryopreservation; hypothermic preservation; ice nucleating effect; recrystallization inhibition; affinity interaction.

samples that failed to survive in control cryostorage conditions (53, 54), whereas in other cases there was no benefit or even impairment of cell viability (38, 64). As far as the mechanism of action is concerned, some researchers have emphasized the ability of these proteins to inhibit recrystallization (4, 6), whereas others champion cell membrane protection (28, 39, 53, 54); yet others propose an ice nucleating effect, a function that is quite inconsistent with the term “AFP” (38, 42). Although it may be difficult to achieve, a critical analysis to clarify the mechanisms of action of AFPs during low-temperature preservation seems to be needed. In recent years, more and more new findings concerning the molecular evolution (16, 20), structural biology (12, 29, 59, 68), and reversible activation (26, 40) of AFPs have been published and these are helpful in elucidating the mechanism of action of these proteins. This paper provides a comprehensive review of the effects of AFPs during low-temperature preservation and discusses possible molecular mechanisms in some detail. It should be mentioned

There are three features of antifreeze proteins (AFPs) that have caused researchers to apply them during low-temperature preservation. First, it is a well-recognized natural phenomenon that they can maintain the supercooled state of body fluids by inhibiting the normal growth habit of ice (11, 52, 69); this property is regardless of the origin of the AFP (14, 19). Second, they have the capacity to inhibit recrystallization, as first identified in in vitro experiments by Knight and co-workers (30, 31). Third, these proteins may serve as plasma membrane protectors at low temperature, as proposed by Rubinsky (55, 56). Contrary to some investigators’ expectations, the results observed when AFPs have been applied to hypothermic storage and cryopreservation are quite complex and even controversial. In some reports, AFPs conferred protection to

Received November 19, 1999; accepted June 16, 2000. This study was supported by a grant from the Natural Science Foundation of China (39900012) and a grant from Zhejiang Natural Science Foundation (399505). 1

0011-2240/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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here that any AFPs that are discussed without specific identification are generally of fish origin. EFFECTS OF AFPs IN HYPOTHERMIC STORAGE

The varied results that have been reported concerning the effect of AFPs during hypothermic storage can be classified into three groups as follows. Group 1 Results in this group show that AFPs possess the ability to protect the cell membrane from hypothermic damage, although this property sometimes seems to be dependent on the type of AFP or its dose. Most of these observations were performed by Rubinsky and his co-workers using 4°C storage. They have reported that a combination of antifreeze glycoproteins (AFGPs) of differing molecular weight, in concentrations of 1– 40 mg/ml, have preserved the structural integrity of the porcine oolemma, although a single ingredient of this combination did not provide protection (56). Three types of AFP, all at 20 mg/ml, ensured that 50% of bovine immature oocytes were able to undergo in vitro maturation and fertilization after storage, whereas the recovery rate was zero in controls (55). The fertilization and early embryo cleavage rates for human oocytes were also increased, in this case by the addition of 1 mg/ml AFP-I or AFP-III, but were reduced as the concentration was raised to 10 mg/ml (28). During 4°C storage of ovine embryos (morula/ blastocyst stages), AFP-I at 1 or 10 mg/ml and AFP-III at 1 mg/ml seemed to be beneficial (51); however, AFP-III at 10 mg/ml lowered the hatching rates, as did 1 mg/ml AFP-I during storage at 0°C (2). In the case of intact rat liver, AFP-III at 15 mg/ml significantly increased bile production and reduced lactate dehydrogenase release (39). Concerning the mechanisms involved in these phenomena, Rubinsky et al. proposed that AFPs block passive ion channels and thereby stabilize transmembrane electrolyte gradients (56, 58).

Group 2 This group of results, from many investigators, describes toxic effects of AFPs. At low temperatures above the freezing point, AFP-III and AFGP appear to be deleterious to spinach thylakoids (25). Upon cooling to 5°C, the motility of ram spermatozoa decreased significantly when the cryoprotectant contained AFP-I or AFGP in concentrations as low as 0.1 ␮g/ml (47). In the case of chimpanzee spermatozoa, AFP-III at 1–100 ␮g/ml also seemed cytotoxic (70). It has been suggested that, both for polar fishes (10) and for cultured frog cells (49), there is little evidence that AFPs have a role in the maintenance of membrane potentials. Freezing of red blood cells in the presence of 40 ␮g/ml AFGP resulted in spicular ice crystals with consequent mechanical stress on the cell membrane (57). At temperatures below the freezing point, all types of AFP aggravated freezing damage in spinach thylakoids (25), as did AFGP (at 10 ␮g/ml and 10 mg/ml) in intact rat heart (64, 71). With cardiomyocytes, AFGPs also exacerbated cell injury by promoting lethal intracellular freezing (42). It has been suggested that the changes in ice crystal morphology that are induced by AFPs, namely the formation of spicular ice, produce shearing effects that account for the destructive action (42, 57). In a series of recent papers on cryosurgery, it has been shown that, at concentrations higher than 5 mg/ml (usually at 10 mg/ml), AFP-I can significantly enhance cellular destruction in cell suspensions (34), in tissues in vitro (33, 35), and in tissues in vivo (50). This is a promising finding for cryosurgery. The damaging effect of AFPs in these studies was also attributed to the mechanical interaction between spicular ice crystals and the cells (33, 34, 35, 50). Group 3 The results classified in this group support a stabilizing effect of AFPs on plasma membranes, but attribute this effect to interactions between AFPs and lipid components of the cell membrane instead of effects on ion channels. It has been reported that, during thermotropic

EFFECTS AND MECHANISMS OF ANTIFREEZE PROTEINS

phase transitions, AFGP at 1–10 mg/ml inhibits the leakage of trapped markers from phospholipid liposomes (23). Incubating human blood platelets with 1 mg/ml AFGP during long-term storage with rapid rewarming abrogated coldinduced activation by preventing lateral-phase separations in the membrane; however, AFP-I had no effect (45, 61). The existence of skintype AFPs, which lack the signal sequences and are expressed abundantly in the external tissues of fishes during cold acclimation, suggests that these proteins may have other functions, apart from reducing ice crystallization in the blood (18, 41). EFFECTS OF AFPs IN CRYOPRESERVATION

The varied results that have been reported following attempts to apply AFPs in cryopreservation can also be classified in three groups, as follows. Group 1 The results in this group suggest that AFPs are effective cryoprotectants. Upon vitrification of immature oocytes and two-cell-stage embryos of mice or pigs, AFGPs at 40 mg/ml produced dramatic improvements in the morphological integrity of the samples, suggesting that these proteins have the ability to inhibit ice formation and to stabilize the plasma membrane (53, 54). Similar results were observed with bovine and ovine embryos at the morula/blastocyst stages (AFP-I and AFGP at 40 mg/ml) (1). Mature mouse oocytes that were cooled rapidly in liquid nitrogen after being exposed at 0°C to 6 mol/L Me 2SO (dimethyl sulfoxide) plus 1 mg/ml AFGP yielded significantly enhanced rates of fertilization and there was less variability of experimental results than in controls. This was probably due to inhibition of devitrification (44). With respect to the two-step method for preservation of ram spermatozoa, AFP-I or AFGP at 10 ␮g/ml increased postthaw motility, although both appeared to be cytotoxic during cooling (47). Similar results have been seen with chimpanzee sperm with 100 ␮g/ml AFP-III (70) and with porcine oocytes at 20 mg/ml AFP-III (8). It has also been reported

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that plant AFGPs improve the recovery of dilated or hatching blastula-stage pig embryos (17). Group 2 The results in this group cast doubt on the benefit of fish AFPs during cryopreservation. In these studies, AFPs failed to show any specific benefits, either in relation to the glass-forming tendency of highly supercooled cryoprotectants (3) or in relation to the survival rates of various cells which include vitrified bovine blastocysts (46), two-step-cryopreserved oyster oocytes (43), and equine embryos (36). AFPs at concentrations between 25 and 1000 ␮g/ml have been reported to damage cells during the cryopreservation of human red blood cells in glycerol (48) and that of hemopoietic cells in Me 2SO (22). It has even been suggested that the inclusion of AFPs during cryopreservation may increase the incidence of intracellular freezing at high subzero temperatures and thereby result in decreased recovery rates (38). Detrimental effects of AFPs have also been described in the cryopreservation of plant cells (62). Group 3 Results in this group conclude that AFPs have both protective and destructive actions, depending on a number of relevant factors (4, 13, 63). At relatively low concentrations (e.g., 5–150 ␮g/ml), AFPs enhanced the survival rate of red blood cells, whereas at millimolar levels, which were much more effective in inhibiting ice recrystallization, these proteins reduced survival rates (4, 6). During the vitrification of rat liver slices, AFP-I at 0.2 mg/ml was beneficial, but the same AFP at 1.0 mg/ml and AFP-III at 0.2 and 1.0 mg/ml had no effect (15). With cryopreserved rice cell suspensions, both the positive effect of recrystallization inhibition and the negative effect of ice nucleation could be observed, depending on the precise combination of a number of factors, including the specific cryopreservation protocol, the concentration of AFP, the composition and concentration of the cryoprotectants, and the physiological features of the cells (62). The dual character of AFP

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JUN-HUI WANG TABLE 1 Properties of Antifreeze Proteins

Temperature condition Cooling Freezing Rewarming

Dual actions Protection of cell membrane Cytotoxicity Inhibition of ice growth Ice nucleation Inhibition of recrystallization Ice nucleation

effects has also been observed in cryomicroscopic and differential scanning calorimetric studies (4, 21, 60), although the concentrations of AFP used varied greatly. INTEGRATION OF THE ACTIONS OF AFPs

To summarize the data concerning the use of AFPs in hypothermic storage, it is concluded that AFPs do indeed interact with plasma membranes, but the action is twofold: membrane protection and cytotoxicity. The balance of these actions in any given situation depends upon several parameters, such as cell surface features, storage temperature, and the type and concentration of AFP. The data concerning the use of AFPs in cryopreservation support the proposal that AFPs do interact with ice, but this action also has two aspects; the inhibition of recrystallization and the nucleation of ice. Table 1 lists the various actions of AFPs, and Fig. 1 demonstrates the relevant elements concerning application of AFPs to cryopreservation. The questions of why and how AFPs possess two opposing functions remains unanswered.

impede further growth of ice (11, 52, 69). Consequently, limited ice growth occurs, predominantly on the basal plane, giving rise to bipyramidal or spicular ice crystals, depending on the concentration of AFP used. More recently, through the use of several structural techniques, including X-ray crystallography, computer simulation, and site-directed mutagenesis, our knowledge of the AFP– ice complementary match has increased considerably. Binding specificity between the single AFP-I helix and the ice has been identified in ultrahigh resolution (27, 59). It is suggested that the entropic effects and van der Waals interactions may play a significant role in binding of AFP-I to ice (7, 9). Globular AFPIII, which lacks the repeating ice-binding residues, has a remarkably flat amphipathic icebinding site where the five hydrogen-bonding atoms fit the two ranks of oxygen atoms in ice (12, 29). This flatness seems to be very important in AFP–ice interplay (68). Configuration studies on AFP-II and AFGP are ongoing (37, 65). With respect to the mechanism of inhibition of recrystallization during the rewarming process, Knight and colleagues (32) have suggested that these proteins bind to the ice crystals at the ice–solution interfaces and thereby prevent the migration of solution and effectively immobilize the ice grain boundaries. Published explanations concerning mechanisms of action of AFPs during low-temperature preservation correlate their biological ef-

MECHANISMS OF ACTION OF AFPs

Inhibition of Ice Growth The well-established ability of AFPs to modify ice growth is known as adsorption inhibition (52, 69). In a system having few ice embryos, the hydrophilic side chains of AFPs bind onto the prism plane (the preferential growth face) of the ice crystals through hydrogen bonds, allowing the exposed hydrophobic side chains of the proteins, with increased surface free energy, to

FIG. 1. Relevant factors in the use of AFPs in cryopreservation.

EFFECTS AND MECHANISMS OF ANTIFREEZE PROTEINS

fects with their ability to modify the structure of ice crystals (33–35, 42, 50, 57, 64, 71). When the dose of AFP is high enough to produce spicular ice crystals and the size of the cells is similar to that of the ice spicules, then AFPs will damage the frozen cells by mechanical distortion of the cell membrane (50, 57) and the formation of intracellular ice (33). On the other hand, when the cells are cryopreserved by vitrification or when the cells are very large relative to the ice spicules and the concentration of AFP is low, then it functions only by inhibiting the recrystallization of ice. However, this simple model cannot explain all the experimental evidence. In some cases, AFPs seem to damage the cells through the promotion of ice growth, even at very low concentrations (48, 62). It is argued in the present paper that AFPs interact not only with the ice crystals but also with cell surfaces and solutes during low-temperature preservation. Such observations should be evaluated to develop other potential explanations of the mechanisms of AFPs. Ice Nucleating Activity Few investigators have attempted to elucidate the origin of ice nucleation in systems containing AFPs. In one paper, it was suggested that the combination of AFP with ice resulted in the stabilization of melting ice embryos, which would lead to increased efficiency as nucleators (5). However, this view is inconsistent with other experiments in which AFPs improved cell viability via the inhibition of recrystallization. In another report, researchers proposed that the hydrophobic interaction of hydrophobic side chains of the AFP–ice complex with the plasma membrane would help water molecules in the membrane to bind with the ice crystals, and this would induce intracellular nucleation and cell destruction (38). However, this view still fails to explain why, in numerous cases, AFPs display beneficial effects and why in acellular systems AFPs also showed ice nucleating activity (24). In this paper, a new model is proposed.

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Basic Description of the Affinity Interaction, Coupled Aggregation Model It is envisaged that the AFP–ice complexes have some affinity interactions with the cell membrane and with many other molecules present in cryopreservation solutions. In some systems, when the intensity of these interactions reaches a certain level, the AFP–ice complexes aggregate, thereby making the ice embryos larger and decreasing the surface free energy. This facilitates the growth of ice and therefore reverses the effect of the AFPs. In other systems, in which the aggregation effect does not occur, AFPs inhibit recrystallization and benefit low-temperature preservation. In some cases, the plasma membrane may participate in and accelerate the aggregation effect. This could occur in two ways: first, the affinity interactions between the AFP–ice complexes and the cell membrane could promote the aggregation of these complexes; second, just as the membrane-mediated aggregation of ice nucleating proteins (INPs) leads to the alignment of the prism planes of ice embryos (66), so the aggregation of AFP–ice around the cell membrane may lead to the alignment of the basal planes of ice embryos. The Source of the Affinity Interactions It has been suggested that, during low-temperature preservation, AFPs can interact with the phospholipid bilayer of the cell membrane (23, 45, 61), possibly by hydrophobic interactions (38). Recent AFP studies indicate much broader interactions between AFPs, the cell membrane, and some solutes. It is reported that AFP-II is homogenous with and evolved from the carbohydrate recognition domains (CRDs) of C-type animal lectins (16, 20), which are able to bind glycoproteins in cell membranes. In addition, contrary to expectation, two thirds of AFP-III ice-binding sites possess a nonpolar solvent-accessible surface (68). Studies on insect AFPs show that, upon cold adaptation, these proteins have a complex interaction with their endogenous activators (26); in vitro experiments have demonstrated that the thermal hys-

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teresis activity of these proteins can be improved or eliminated by a number of lowmolecular-mass compounds (40). Evidence from some bacterial AFPs containing carbohydrate moieties indicates that removal of these carbohydrates results in the disappearance of the ice nucleating effect (67). In conclusion, it has been shown that AFPs not only specifically interact with ice but also compatibly interact with numerous other molecules present in the solutions and in the cell membrane. The Intensity of the Affinity Interactions The intensity of the affinity interactions must be affected by those factors that alter the final results of low-temperature preservation in the presence of AFPs, that is to say, the different parameters illustrated in Fig. 1. These include the concentration and type of AFP, the composition and concentration of cryoprotectant, the cooling and rewarming rates, and the cell surface features, all of which alter the intensity of the affinity interactions and affect the possibility of the aggregation effect occurring. As reviewed in this paper, samples that failed to survive or that had very low recovery rates in control cryopreservation systems sometimes showed remarkable improvement after inclusion of AFPs (17, 44, 53, 54). It is suspected that, under normal conditions, the cell surface of these systems may be relatively polar and therefore easily damaged by ice crystals, whereas in the presence of AFPs, exposure of nonpolar side chains of AFP–ice results in an inefficient interaction between ice and cell membrane. Because there is then less likelihood of membranemediated ice nucleation, a beneficial effect of AFP is observed. Obviously, the model presented in this paper is speculative. Many authors believe that it is very easy to introduce contaminating ice nucleators into AFP samples, making it difficult to be certain whether the ice nucleating action is produced by AFPs or by contaminants. In addition, different types of AFPs may have differing ice nucleating activities. Some authors even speculate that AFPs have the ability to distribute

water molecules in an ice-like fashion to promote the growth of ice. CONCLUSION AND PROSPECTS

AFPs have a dual effect in low-temperature storage, with regard both to cell membranes and to ice growth. Considering some of the newer findings concerning AFPs and INPs, it has been possible to propose an integrated model for the action of AFPs during cryopreservation. It is suggested that affinity interactions, which vary between different cryopreservation systems, control the occurrence of the aggregation effect. In the absence of the aggregation effect, AFPs act as recrystallization inhibitors and may alleviate cryoinjury; on the other hand, when aggregation occurs, the AFPs serve as ice nucleators and lead to cell membrane damage. The exact mechanism of ice nucleation by AFPs remains unresolved. Fish AFPs have been studied intensively, but there is little published work on the use of insect and plant AFPs in hypothermic storage and cryopreservation. Current work is investigating these two areas. The dual character of fish AFPs during low-temperature preservation implies that transfer of the genes for these proteins into freezing-intolerant terrestrial plants or animals may prove to be complicated, as may the studies to develop the use of AFPs in cryosurgery. Nevertheless, AFPs promise to be useful both in theoretical and in applied cryobiology. ACKNOWLEDGMENTS The author is sincerely grateful to the referees for valuable comments and suggestions, to the editor-in-chief for encouragement, and to Professor Kai-Zhi Wang for helpful advice on the English language presentation of the manuscript. REFERENCES 1. Arav, A., Ramsbottom, G., Baguisi, A., Rubinsky, B., Roche, J. F., and Boland, M. P. Vitrification of bovine and ovine embryos with the MDS technique and antifreeze proteins. Cryobiology 30, 621– 622 (1993). 2. Baguisi, A., Arav, A., Crosby, T. F., Roche, J. F., and Boland, M. P. Hypothermic storage of sheep embryos with antifreeze proteins: Development in vitro and in vivo. Theriogenology 48, 1017–1024 (1997).

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