Fact and Fantasy

John H. Crowe* Abstract

Trehalose is a disaccharide of glucose that is found at high concentrations in a wide variety of organisms that naturally survive drying in nature. Many years ago we reported that this molecule has the remarkable ability to stabilize membranes and proteins in the dry state. A mechanism for the stabilization rapidly emerged, and it was sufficiently attractive that a myth grew up about trehalose as a universal protectant and chemical chaperone. Many of the claims in this regard can be explained by what is now known about the physical properties of this interesting sugar. It is emerging that these properties may make it unusually useful in stabilizing intact cells in the dry state.

Sugars and Stabilization of Biological Materials

We reported two decades ago that biomolecules and molecular assemblages such as membranes and proteins can be stabilized in the dry state in the presence of a sugar found at high concentrations in many anhydrobiotic organisms, trehalose.1 We also showed that trehalose was clearly superior to other sugars in this regard.2 This effect seemed so clear it quickly led to wide-spread, and often uncritical, use of the sugar for preservation and other purposes. In fact, an array of applications for trehalose have been reported, ranging from stabilization of vaccines and liposomes to hypothermic storage of human organs.3 Other workers showed that it might even be efficacious in treatment of dry eye syndrome or dry skin in humans.4'5 Trehalose is prominendy listed as an ingredient in cosmetics, apparendy because it is reputed to inhibit oxidation of certain fatty acids in vitro that might be related to body odor.6 Trehalose has been shown by several groups to suppress free radical damage, protect against anoxia, inhibit dental caries, enhance ethanol production during fermentation, stabilize the flavor in foods, and to protect plants against physical stress.7"13 According to one group, trehalose inhibits bone resorption in ovariectomized mice, apparendy by suppressing osteoclast differentiation; the suggestion followed that trehalose might be used to treat osteoporosis in humans.14'15 More re-cendy, Tanaka et al16 reported that trehalose could be used to inhibit the protein aggregation associated with Huntington's disease in vivo in a rat model for this disease. That report that has already led to an unorthodox clinical trial in humans.17

A myth has grown up about trehalose and its properties, as a result of which it is being applied, sometimes rather uncritically, to a myriad of biological and clinical problems. Thus, we are making special efforts in the literature to clarify the properties of trehalose that make it useful for stabilization of biomaterials and to dispel the most misleading aspects of this myth.

*Corresponding Author: Dr. John H. Crowe—Section of Molecular and Cellular Biology, University of California, Davis, California 95616, U.S.A. Email: [email protected]

Origins of the Trehalose Myth

We recently reviewed the history of this field (see ref. 3) and provide only a brief summary here. The key observations were: (1) The first model membrane investigated was sarcoplasmic reticulum, isolated from lobster muscle (reviewed in ref. 18). We found that trehalose was without question superior to all other sugars tested at preserving these membranes during drying. However, we later obtained evidence that these SR membranes have a mechanism for translocating trehalose across the bilayer. We suggest that other sugars such as sucrose might preserve the membranes at concentrations similar to those seen with trehalose if they had access to the aqueous interior. (2) Initial studies with liposomes, from the mid-1980s (reviewed in ref. 19), were done with a phosphoplipid with low Tm. When the liposomes were freeze dried with trehalose and rehydrated, the vesicles were seen to be intact, and nearly 100% of the trapped solute was retained. It quickly emerged that stabilization of these liposomes, and other vesicles prepared from low melting point lipids, had two requirements, as illustrated in Figure 1: (a) inhibition of fusion between the dry vesicles; and (b) depression of Tm in the dry state. In the hydrated state, Tm for egg PC is about -1°C and rises to about + 70°C when it is dried without trehalose. In the presence of trehalose, Tm is depressed in the dry state to - 20°C. Thus, the lipid remains in liquid crystalline phase in the dry state, and phase transitions are not seen during

Hydrated POPC

above Tm

Heat above Tm

Heat above Tm

Heat above Tm

Coot

belcw Tm m

Figure 1. Mechanism for stabilization of phospholipid bilayers in the dry state. Adapted from reference 18.

rehydration. The significance of this phase transition during rehydration is that, when phospholipids pass through such transitions, the bilayer becomes transiendy leaky. The physical basis for this leakiness has recendy been investigated in some detail.20 These effects were reported first for trehalose (reviewed in ref. 19). When we compared the effects of other sugars and polymers on the preservation, we found that, with vesicles made from lipids with low Tm, trehalose appeared to be significantly superior to the best of the additives tested. Oligosaccharides larger than trisaccharides did not work at all.21 Other sugars, particularly disaccharides, did provide good stabilization of egg PC vesicles in the dry state, but much higher concentrations than trehalose were required, at least according to initial reports. However, as freeze-drying technology improved, the differences between disaccharides tended to disappear, and the myth eventually got modified to encompass disaccharides in general. Nevertheless, the observation that trehalose was significandy more effective at low concentrations under suboptimal conditions for freeze drying requires explanation, which we provide later. (3) At first it appeared that the ability to preserve liposomes in the dry state is restricted to disaccharides. Subsequendy, we found this is not the case. For example, DPPC is a lipid with saturated acyl chains and thus an elevated Tm (41 °C). When it is dried without trehalose Tm rises to about 110°C; with trehalose present Tm rises to about 65°C (reviewed in ref. 22). Thus, DPPC is in gel phase at all stages of the freeze-drying and rehydration process, and one would expect that inhibition of fusion might be sufficient for the stabilization. In other words, any inert solute that would separate the vesicles in the dry state and thus prevent aggregation and fusion should stabilize the dry vesicles. That appears to be the case; a high molecular weight (450,000) HES has no effect on Tm in dry DPPC, but preserves the vesicles, nevertheless.

The Mechanism of Depression ofTm

The Mechanism of Depression ofTm has received a great deal of attention since the discovery of this effect.23 Three main hypotheses have emerged: The water replacement hypothesis suggests that sugars can replace water molecules by forming hydrogen binds with polar residues, thereby stabilizing the structure in the absence of water.2,2 26 The water entrapment hypothesis suggests that sugars concentrate water near surfaces, thereby preserving its salvation.27"29 The vitrification hypothesis suggests that the sugars form amorphous glasses, thus reducing structural fluctuations.30"31

A consensus has emerged that these three mechanisms are not mutually exclusive (reviewed in ref. 3). Vitrification may occur simultaneously with direct interactions between the sugar and polar residues. Direct interaction, on the other hand, has been demonstrated by a wide variety of physical techniques, including infrared spectroscopy, NMR, and X-ray.23,3 36

Theoretical analyses have contributed gready to this field in recent years. Chandrasekhar and Gaber37 and Rudolph et al38 in the earliest studies, showed that trehalose can form energetically stable conformations with phospholipids, binding three adjacent phospholipids in the dry state. Similarly, trehalose-protein interactions have been studied by simulations, with similar conclusions.28,29 More recently, Sum et al39 showed by molecular simulations that the sugars adapt molecular conformations that permit them to fit onto the surface topology of the bilayer through hydrogen bonds. The sugars interact with up to three adjacent phospholipids. Pereira et al4 produced complementary results from molecular dynamics simulations, with comparable conclusions.

Trehalose Stabilizes Microdomains in Membranes

Phase separation is segregation of membrane components in the plane of the bilayer. Although there are lingering doubts about whether or not phase separated domains in native membranes are real (see refs. 41-43) or artifacts (see refs. 44-46), there is abundant evidence that these domains, known as "rafts", are involved in such processes (among others) as signaling, endocytosi, and viral assembly.47"51 Although several forces are involved, one of the main driving forces for phase separation is the hydrophobic mismatch, which arises from a difference in membrane thickness between two species within a bilayer, such as a protein and a lipid or a lipid and a lipid.52,53 The differences in thickness lead to exposure of hydrophobic residues to water and, consequendy, to a decrease in entropy of the system resulting from ordering of the water. Thus, the assembly of components of similar thickness into relatively homogeneous domains is entropi-cally driven. The net increase in entropy driving the process is contributed by water.

Phase separated domains in lipid bilayers are becoming increasingly well understood (see ref. 54 for a recent review). Thus, we have investigated whether the domains can be maintained in freeze-dried liposomes. DLPC (Tm = 0°C) and DSPC (Tm = 50°C) are well known to undergo complete phase separation in the fully hydrated state.55,56 When these liposomes were dried, the two lipids underwent extensive mixing. In samples dried with trehalose, by contrast, the DLPC transition is depressed to about -20°C, and the DSPC transition increases by about 10°C and becomes more cooperative, suggesting that it is more like pure DSPC. Thus, the phase separation—and the domain structure—are maintained by the trehalose in the dry state. Other pairs of lipids that phase separate when fully hydrated give similar results.

We propose that trehalose maintains phase separation in this mixture of lipids in the dry state by the following mechanism.36 The DLPC fraction, with its low Tm in the hydrated state, might be expected to behave like unsaturated lipids, in that Tm in the dry state is reduced to a minimal and stable value immediately after drying with trehalose, regardless of the thermal history. That appears to be the case. The DSPC fraction, by contrast, would be expected to behave like DPPC, as described earlier. DSPC is in gel phase in the hydrated state at room temperature, and it remains in gel phase when it is dried with trehalose. In other words, we are proposing that by maintaining one of the lipids in liquid crystalline phase during drying, while the other remains in gel phase, trehalose maintains the phase separation (Fig. 2). We suggest that this is the fundamental mechanism by which trehalose maintains phase separated domains in membranes drying.

- Trehalose

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