Subzero Temperature Adaptations, Antifreeze Proteins

John Duman

Martin J. Gillen Professor of Biological Sciences
Ph.D., University of California, San Diego (Scripps Institution of Oceanography)

Research concerns the physiological and biochemical adaptations of poikilothermic organisms to subzero temperature. Most studies are on insects, but other organisms (including plants, spiders and other terrestrial invertebrates) are also under investigation. These organisms adapt to subzero winter temperatures by either becoming freeze tolerant (able to survive extracellular freezing) or freeze avoiding. Freeze avoiding species generally produce antifreezes, such as polyols (glycerol, etc) and/or antifreeze proteins (AFPs). We have been particularly concerned with the AFPs, and our studies have ranged from investigations of the function of the proteins, to hormonal and environmental cues controlling their production, to protein chemistry and molecular biology designed to determine the structure – antifreeze function relationships of the proteins. Studies with freeze tolerant organisms have concentrated on ice nucleating proteins which function to induce ice formation in the extracellular fluid at high subzero temperature, and also on their AFPs. Antifreeze proteins function in certain freeze tolerant organisms as cryoprotectants to inhibit the damage resulting from freezing of body water. The mechanisms of this process are under investigation, but include recrystallization inhibition (control of ice crystal size) and inhibition of the generally lethal spread of extracellular ice into the cytoplasm. Recently, a new type of thermal hysteresis factor/antifreeze that is a glycolipid has been characterized. The antifreeze glycolipid has levels of antifreeze activity comparable to insect AFPs and is assocciated mainly with cell membrances where it may inhibit intracellular ice formation. Current research includes: (1) structure/function relationships of insect and plant antifreeze proteins and glycolipids; (2) the cloning and expression of antifreeze protein genes; (3) cryoprotection mechanisms of antifreeze proteins and glycolipids in freeze tolerance; (4) applied studies on (a) the potential uses of antifreeze proteins and glycolipids in agriculture (i.e., transgenic plants which produce insect antifreeze proteins) and (b) the cryopreservation of biomedically important tissues and organs; and (5) studies of antifreeze proteins and related adaptations in Alaskan insects, including proteomic and other studies of how the beetle Cucujus clavipes deep supercools to -100 degrees C. Within the broad theme of low temperature adaptations a wide variety of student projects, ranging from physiological ecology to protein biochemistry, can be accommodated.

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Selected Recent Publications:

Bennett, V. A., Sformo, T., Walters, K., Toien, O., Jeannet, K., Hochstrasser, R., Pan, Q., Serianni, A.S., Barnes, B.M., and Duman, J.G. (2005) Comparative overwintering physiology of Alaska and Indiana populations of the beetle Cucujus clavipes (Fabricus): Roles of antifreeze proteins, polyols, dehydration, and diapause. Journal of Experimental Biology 208: 4467-4477.

Wang, L. and Duman, J.G. (2006) A thaumatin-like protein from larvae of the beetle Dendroides canadensis enhances the activity of antifreeze proteins. Biochemistry 45: 1278-1284.

Nicodemus, J., O’Tousa, J.E., and Duman, J.G. (2006) Expression of a beetle, Dendroides canadensis, antifreeze protein in Drosophila melanogaster. Journal of Insect Physiology 52: 888-896.

Walters, K.R., Sformo, T., Barnes, B.M., and Duman, J.G. (2009) Freeze tolerance of an Arctic Alaska stonefly. Journal of Experimental Biology 212: 305-312.

Sformo, T., Kohl, F., McIntyre, J., Kerr, P., Duman, J. G., and Barnes, B. M. (2009) Simultaneous freeze tolerance and avoidance in individual fungus gnats, Exechia migratoria. Journal of Comparative Physiology B 179: 897-902.

Walters, K. R., Pan, Q., Serianni, A. S., and Duman, J., G. (2009) Cryoprotectant biosynthesis and selective accumulation of threitol in the freeze tolerant beetle, Upis ceramboides. Journal of Biological Chemistry 284: 16,822-16,831.

Walters, K.R., Serianni, A.S., Sformo, T., Barnes, B.M., Duman, J.G. (2009) A novel thermal hysteresis-producing xylomannan antifreeze in a freeze tolerant Alaskan beetle. Proceedings of the National Academy of Science 106: 20210-20215.

Duman, J. G., Walters, K. R., Sformo, T., Carrasco, M. A., Nickell, P. and Barnes, B. M. (2010) Antifreeze and ice nucleator proteins. In “Low Temperature Biology of Insects” (D. Denlinger and R. E. Lee, eds) Cambridge University Press, Cambridge, UK. pp 59-90.

Sformo, T., Walters, K., Jeannet, K., McIntyre, J., Wowk, B., Fahy, G., Barnes, B.M., Duman, J.G. (2010) Deep Supercooling, vitrification, and limited survival to -100 degrees C in larvae of the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujuidae). Journal of Experimental Biology 213: 502-509.

Lin, X., O'Tousa, J.E., and Duman, J.G. Expression of two self-enhancing antifreeze proteins from the beetle Dendroides canadensis in Drosophila melanogaster. (2010) Journal of Insect Physiology 56: 341-349

Walters, K.R. Serianni, A.S., Voituron, Y., Sformo, T., Barnes, B.M., and Duman, J.G. (2011) A thermal hysteresis-producing xylomannan glycolipid antifreeze associated with cold-tolerance is found in diverse taxa. Journal of Comparative Physiology B, 181:631-640.

Brockbank, K.G.M., Campbell, L.H., Brockbank, M.C.G., and Duman, J.G. (2011) Lessons learned from nature for preservation of mammalian cells, tissues and organs. In Vitro Biology – Animal, 47: 210-217.

Lin, X., Wisniewski, M. E. and Duman, J.G. (2011) Expression of two self-enhancing antifreeze proteins from the beetle Dendroides canadensis in Arabidopsis thaliana. Plant Molecular Biology Reporter, 29: 802-813.

Sformo, T., McIntyre, J., Walters, K.R., Barnes, B.M. and Duman, J.G. (2011) Probability of freezing in the freeze avoiding beetle larvae Cucujus clavipes puniceus (Coleoptera, Cucujidae) from Interior Alaska. Journal of Insect Physiology. 57: 1170-1177.

Carrasco, M.A. and Duman, J.G. (2011) A cross-species compendium of proteins related to cold stress identified by bioinformatic approaches. Journal of Insect Physiology 57:1127-1135.

Carrasco, M.A., Buechler, S., Arnold, R., Sformo, T., Barnes, B.M., Duman, J.G.(2011) Elucidating the biochemical overwintering adaptations of larvae of the Alaskan beetle Cucujus clavipes puniceus, a non-model organism, via high throughput proteomics. Journal of Proteome Research 10: 4634-4646.

Carrasco, M.A., Buechler, S., Arnold, R., Sformo, T., Barnes, B.M., Duman, J.G.Investigating the deep supercooling ability of an Alaskan beetle, Cucujus clavipes puniceus, via high throughput proteomics. Journal of Proteomics (in press).

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