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Heat-Stable Antifreeze Protein from Grass

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Heat-Stable Antifreeze Protein from Grass brief communications relatively straightforward mechanisms, for Shear, W.) 319–363 (Stanford Univ. Press, Palo Alto, CA, 1986). insect Dendroides canadensis thermal- example by the modification of particular 6. Witt, P. N., Reed, C. & Peakall, D. B. A Spider’s Web (Springer, hysteresis protein, which does7. 14,15 New York, 1968). receptors . Hymenoepimecis’s manipula- 7. Fincke, O., Higgins, L. & Rojas, E. J. Arachnol. 18, 321–329 Our Lolium antifreeze protein had a sig- tion of its spider host is probably the most (1990). nificantly higher specific activity in an ice- 8 finely directed alteration of behaviour ever 8. Griswold, C. E., Coddington, J. A., Hormiga, G. & recrystallization inhibition assay than attributed to an insect parasitoid. Scharff, N. Zool. J. Linn. Soc. 123, 1–99 (1998). other antifreeze proteins. Growth of ice 9. Wenzel, J. W. Annu. Rev. Ecol. Syst. 23, 361–381 (1992). William G. Eberhard 10.Holmes, J. C. & Bethel, W. M. in Behavioural Aspects of Parasite crystals in 30% sucrose solution was com- Smithsonian Tropical Research Institute, and Transmission (eds Channing, E. U. & Wright, C. A.) 123–149 pletely inhibited at AFP concentrations 1 Escuela de Biología, Universidad de Costa Rica, (Academic, London, 1972). below 10 mg ml 1 (Fig. 1c,d), which is at Ciudad Universitaria, Costa Rica 11.Barnard, C. J. & Behnke, J. M. Parasitism and Host Behaviour least 200 times less in molar terms than the (Taylor & Francis, London, 1990). e-mail: [email protected] 12.Toft, C. A., Aeschlimann, A. & Bolis, L. Parasite–Host type III AFP from ocean pout (Macro- 1. Eberhard, W. G. J. Hymen. Res. (in the press). Associations (Oxford Univ. Press, New York, 1991). zoarces americanus). 2. Eberhard, W. G. J. Arachnol. 18, 205–234 (1990). 13.Godfray, H. C. J. Parasitoids: Behavioral and Evolutionary In contrast, Lolium AFP shows a low 3. Tilquin, A. La Toile Géometrique des Araignées (Presses Univ. Ecology (Princeton Univ. Press, Princeton, NJ, 1994). France, Paris, 1942). 14.Jenni, L., Molyneux, D. H., Livesey, J. L. & Galun, R. Nature thermal hysteresis (the lowering of the tem- 4. Eberhard, W. G. Evolution 36, 1067–1095 (1982). 283, 383–385 (1980). perature at which ice forms on cooling 5. Coddington, J. A. in Spiders: Webs, Behavior and Evolution (ed. 15. Wickler, W. Z. Tierpsychologie 42, 200–214 (1976). while the melting temperature remains unaltered9), with the highest measurable value being 0.1 °C in water and 0.45 °C in 30% sucrose; these values are much lower Phytochemistry ab than the 1.0–1.5 °C reported for fish AFP10 and the 5–6 °C reported for insect pro- Heat-stable antifreeze teins11, although these too are increased by protein from grass sucrose12. Mechanisms previously proposed to e have discovered an antifreeze pro- explain how AFPs work all imply some cor- 1 tein in an overwintering perennial [0001] [1120] relation between thermal hysteresis effects Wryegrass, Lolium perenne. The pro- and recrystallization inhibition13. Our dis- tein is stable at 100 °C and although it is a [1210] covery that there is no correlation between less effective antifreeze than proteins found [1210] these relative activities of the antifreeze pro- in antarctic fish and insects, it is better tein from L. perenne raises questions about [2110] at preventing ice recrystallization. This cd the nature of the AFP mechanism and indi- property enables grasses to tolerate ice for- cates that different classes of AFP may inter- mation in their tissues without being act with ice in different ways. We propose damaged, suggesting that the control of that the thermal hysteresis activity of the ice-crystal growth rather than the preven- grass protein is unlikely to serve an impor- tion of freezing may have evolved to be the tant protective function at the very low tem- critical factor in their survival at very low Figure 1 Lolium antifreeze protein (AFP) binding to ice and its peratures survived by overwintering grasses, temperatures. effect on ice recrystallization. Lolium AFP binds specifically to an whereas its capacity to control the growth of Frost-tolerant plants undergo a process ice-crystal surface with six-fold symmetry. a,b, Ice-etching deter- ice crystals may protect it against damage to of cold acclimation2,3, during which peren- mination of the binding planes using the hemisphere technique5: the plant cellular structure. nial grasses accumulate a boiling-tolerant a, three elongated patches positioned on the primary prism plane; Chris Sidebottom, Sarah Buckley, protein that inhibits ice recrystallization. and, b, the planes symmetrically arranged around the crystal’s c- Paul Pudney, Sarah Twigg, Carl Jarman, We extracted the protein responsible for axis. c,d, Influence of Lolium AFP on ice recrystallization. The Chris Holt, Julia Telford, this activity from cold acclimated leaves of recrystallization inhibition assay shows crystal growth after 60 min Andrew McArthur, Dawn Worrall*, L. perenne and cloned its complementary at 16 °C; Lolium perenne (d) inhibits recrystallization of ice in Rod Hubbard*, Peter Lillford DNA by using the polymerase chain reac- dilute concentrations relative to growth with the 30% sucrose Unilever Research, Colworth House, Sharnbrook, tion4. We found that it had an open reading control (c). Scale bar, 50 mm. Bedford MK44 1LQ, UK frame encoding a protein of 118 amino e-mail: [email protected] acids (GenBank accession number, able to hydrogen-bond with an ice surface. *Plant Laboratory, Biology Department, AJ277399) and relative molecular mass Growth of a single ice-crystal hemisphere University of York, York YO1 5YW, UK 11.765K, with six potential N-glycosylation from a dilute solution of the protein, and 1. Feeney, R. E. & Yeh, Y. Food Technol. 47, 82–88 (1993). sites containing the conserved N-X-S/T gly- subsequent surface-etching of the ice hemi- 2. Thomashow, M. F. Adv. Genet. 28, 99–131 (1990). cosylation motif. sphere5, produced a distinctive pattern with 3. Guy, C. L. Annu. Rev. Plant Physiol. 41, 187–223 (1990). Although this boiling-tolerant antifreeze six-fold symmetry, demonstrating that the 4. Frohman, M. A., Dush, M. K. & Martin, G. R. Proc. Natl Acad. Sci. USA 85, 8998–9002 (1988). protein (AFP) belongs to a new class of protein was specifically binding to ice on 5. Knight, C. A., Cheng, C. & DeVries, A. L. Biophys. J. 59, plant proteins and shares no lengthy the primary prism plane (Fig. 1a,b). 409–418 (1991). sequence homology with any other AFP or The Fourier-transform infrared spec- 6. Arrondo, J. L. R., Young, N. M. & Mantsch, H. H. Biochim. Biophys. Acta 952, 261–268 (1988). protein sequence, some of its properties fit trum of this grass AFP in solution at room 7. Li, N., Kendrick, B. S., Manning, M. C., Carpenter, J. F. & with the general pattern for AFPs. It is very temperature revealed a high solvent- Duman, J. G. Arch. Biochem. Biophys. 360, 25–32 (1998). hydrophilic, being rich in asparagine exposed b-sheet content which may be 8. Worrall, D. et al. Science 282, 115–117 (1998). (25%), valine (16%), serine (15%) and exposed at the ice-binding surface6, as pro- 9. Knight, C. A., DeVries, A. L. & Oolman, L. D. Nature 308, 295–296 (1984). threonine (10%), and having very few posed for several other antifreeze proteins, 10.Duman, J. G., Wu, D. W., Olsen, T. M., Urrutia, M. & amino acids with aromatic or hydrophobic including that from carrot and types II and Tursman, D. Adv. Low-Temp. Biol. 2, 131–182 (1993). side chains. The primary structure has a III from fish. The spectrum was the same in 11.Graham, L. A., Liou, Y.-C., Walker, V. K. & Davies, P. L. Nature series of highly conserved, 7-amino-acid the presence of ice, suggesting that the con- 388, 727–728 (1997). 12.Li, N., Andorfer, C. A. & Duman, J. G. J. Exp. Biol. 201, repeat sequences with regularly spaced formation of the Lolium AFP does not 2243–2251 (1998). serine and threonine residues that may be change on binding to ice, unlike that of the 13.Hall, D. G & Lips, A. Langmuir 15, 1905–1912 (1999). © 2000 Macmillan Magazines Ltd 256 NATURE | VOL 406 | 20 JULY 2000 | www.nature.com.
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