PROJECT TITLE NUMBER: IOS 1025929
Organisms that are exposed to subzero temperature adapt by becoming either freeze tolerant (they survive being frozen) or they must become freeze avoiding to prevent freezing. Structurally diverse antifreeze proteins (AFPs) have evolved in many different organisms: animals, plants, bacteria, fungi, etc., but insect AFPs are arguably the most active. AFPs inhibit freezing by binding to the surface of ice crystals and/or ice nucleating surfaces, thereby reventing water molecules from joining the crystal surface. Consequently, AFPs lower the freezing point of an aqueous solution, but do not change the melting point, producing the thermal hysteresis (TH, difference between the freezing and melting points) characteristic of their presence. Previously, only antifreeze proteins were known to have this activity. Intellectual Merit. Recently, we identified novel glycolipids with TH equal to that of insect AFPs. These new antifreeze glycolipids (AFGLs) were found in several cold tolerant insects (both freeze tolerant and freeze avoiding), a frog, a fish, and a freeze tolerant plant. Since thermal hysteresis has previously been identified only in proteins, this novel discovery has the potential to transform our ideas on how organisms adapt to subzero temperatures. AFP function has been best studied in freeze avoiding species where they function to prevent freezing by blocking inoculative freezing across the surface from external ice and by inhibiting ice nucleators in body fluids. One species that we study, an Alaskan beetle, Cucujus clavipes, produces typical beetle type AFPs that assist them to deep supercool, so that they do not freeze even if taken to ?150oC. At ~-70oC the body water vitrifies, turns to glass, but does not freeze. C. clavipes is one of the species that produces AFGLs. We propose to continue studies of this interesting insect to determine the potential synergy in physiological function of these antifreezes. We will also investigate the structure and physiological function of AFGLs in two freeze tolerant insects, (Upis ceramboides) from Alaska (freeze tolerant to ~-60oC) and (Tipula trivittata) from Indiana (freeze tolerant to ~-28oC), and in a freeze tolerant plant, the bittersweet nightshade Solanum dulcamara from Indiana. The function of TH-antifreezes (AFPs or the AFGL) in freeze tolerant species is not well understood. Recall that these species have evolved to freeze and survive, so why have antifreeze? One possibility is that, since these organisms generally only survive freezing of their extracellular water, the AFGLs may function to prevent the lethal spread of ice from the extra- to the intra-cellular water. In fact, this may be the case since most of the AFGL in these species is associated with cell membranes, perfectly situated for this function. The primary scientific goal of this study is to (1) determine the structure of the AFGLs, and (2) to identify their physiological functions in both freeze tolerant and freeze avoiding organisms. The broader impacts of this study are three-fold: (1) potential cryopreservation of biomedical materials, (2) potential improved crop and horticultural plant cold tolerance and (3) a positive effect on biological education. AFPs, and now the novel AFGLs, have possible applications in the cryopreservation of cells, tissues and organs. AFGLs may provide freeze protection to cells, permitting them to be more easily freeze-preserved. AFGLs may also permit subzero storage of materials in the unfrozen state, mimicking their function in the deep supercooling C. clavipes from Alaska. There could also be applications in agriculture resulting in more cold tolerant plants. Students at the high school, undergraduate, PhD and post-doctoral levels will be directly involved in this study, thereby receiving interdisciplinary training ranging from field biology and physiological ecology to biochemistry and molecular biology. In addition, elucidation of these adaptations has the potential to attract new students and practicing scientists from diverse backgrounds. In this era of increased specialization when biochemists and ecologists, biologists and physicists or chemists seem to have few common interests, such studies can foster considerable interdisciplinary understanding and cooperation. Our recent initial publication of AFGLs has received widespread general attention, ranging from the NY Times to Nature magazine. Also, we will provide a service to cold tolerance researchers by screening their organisms for AFGLs.
Antifreeze glycolipids (AFGLs) are a newly discovered class of naturally occurring antifreezes similar in activity to the well-known antifreeze proteins. The goals of this study were to elucidate the structure and physiological function of the AFGLs. Intellectual Merit. Physiological Function. AFGLs are primarily present on the cell membranes of freeze tolerant organisms (those able to survive freezing) such as certain insects, frogs and plants. In general, these organisms are only able to survive ice formation in their extracellular spaces (outside the cells), as intracellular freezing of the cytoplasm is usually lethal. We demonstrated that the AFGLs function primarily to prevent the spread of the tolerated extracellular ice into the cytoplasm, thereby protecting the cells from lethal intracellular ice formation. Two sets of experiments done on cells from non-cold tolerant organisms demonstrated this especially well. (1) Cells of summer geraniums, a very cold sensitive plant, treated with AFGL and exposed to decreasing subzero temperatures in the presence of ice froze at a significantly lower temperature (in one experiment below -20C) than control cells without AFGL (-5C or above). (2) Similar experiments using mammalian vascular smooth muscle cells (rat AK10) exposed to subzero temperatures showed similar results, demonstrating much improved viability of the cells treated with AFGL compared to untreated cells or cells treated with a standard cell cryoprotectant, DMSO. This improved viability manifested both immediately following thawing of the cells and after three days of growth as a monolayer. In addition to their presence on cell membranes, lesser amounts of AFGLs are generally present free in the extracellular fluid where they function primarily to prevent recrystallization of ice, a process whereby there is a net migration of water molecules from the surface of smaller ice crystals to larger crystals, because of the lower surface free energy on the surface of the larger crystals. (Recrystallization is the process that leads to large ice crystals in ice cream that is left in the freezer for some time.) Because recrystallization is damaging to tissue, the recrystallization inhibition activity of AFGLs, even at very low concentrations, helps to avoid freeze damage. Structure. At the initiation of this study we suspected that the basic core of the AFGLs was a repeating unit of a disaccharide consisting of mannose and xylose with an unusual ß 1→4 linkage [ß-D-Manp-(1→4)-ß-D-Xylp (ßManp-ßXylp)] attached to lipid. This study confirmed this core structure. However, other goals concerning the AFGL structure were not reached. These include: (a) whether the core disaccharide unit is linear or if it contains branchpoints, and (b) the nature of the lipid components and how they are attached to the saccharide. Other Findings. (1) With colleagues at the University of Alaska, Fairbanks, we showed that wood frogs, Lithobates (Rana) sylvatica, from Interior Alaska have AFGLs. The Alaskan frogs are freeze tolerant to much lower temperatures (~-20C) than those from northern USA and southern Canada (~-6 to -8C). (2) Synergistic self-enhancement of antifreeze activity (thermal hysteresis) between AFGLs and antifreeze proteins from larvae of the beetle Dendroides canadensis (DAFPs) was identified. When combined the activities of DAFP-2 from the beetle and AFGLs were enhanced by approximately 1.5-fold for the DAFP and by 3.0-fold for the AFGL. This suggests that other activities of the antifreezes will be similarly enhanced. Broader Impacts. Findings from this study suggest that AFGLs may be of value in cryopreservation of biomedical materials and perhaps as components of sprays for protection of crop plants from frost damage.
