A mutation to a fish ice-binding protein synthesized in transgenic Caenorhabditis elegans modulates its cold tolerance.

A cryoprotectant known as ice-binding protein (IBP) is thought to facilitate the cold survival of plants, insects, and fungi. Here, we prepared a genetically modified Caenorhabditis elegans strain to synthesize fish-derived IBPs in its body wall muscles and examined whether the antifreeze activity modification of this IBP by point mutation affects the cold tolerance of this worm. We chose a 65-residue IBP identified from notched-fin eelpout, for which the replacement of the 20th alanine residue (A20) modifies its antifreeze activity. These mutant proteins are denoted A20L, A20G, A20T, A20V, and A20I along with the wild-type (WT) protein. We evaluated the survival rate (%) of the transgenic C. elegans that synthesized each IBP mutant following 24 h of preservation at -5, +2, and +5 °C. Significantly, a dramatic improvement in the survival rate was detected for the worms synthesizing the activity-enhanced mutants (A20T and A20I), especially at +2 °C. In contrast, the rate was not improved by the expression of the defective mutants (A20L, A20G, WT and A20V). The survival rate (%) probably correlates with the antifreeze activity of the IBP. These data suggest that IBP protects the cell membrane by employing its ice-binding mechanism, which ultimately improves the cold tolerance of an IBP-containing animal.

Expression of Ice-Binding Proteins in Caenorhabditis elegans Improves the Survival Rate upon Cold Shock and during Freezing.

Ice-binding proteins (IBPs) are capable of binding ice crystals and inhibiting their growth at freezing temperatures. IBPs are also thought to stabilize the cell membrane at non-freezing temperatures near 0 °C. These two effects have been assumed to reduce cold- and freezing-induced damage to cells and tissues. However, knowledge regarding the effects of IBP on the living animals is limited. Here, we characterized the relationship between the IBP effects and the physiological role by using the nematode Caenorhabditis elegans. The expression of fish (NfeIBPs)- and fungus-derived IBPs (AnpIBPs and TisIBP8) in C. elegans improved its survival rate during exposure to 0 and -2 °C (cold shock) and -5 °C (freezing). The observed cold tolerance of C. elegans after cold shock is attributable to the stabilization of cell-membrane lipids with IBPs, and the freezing tolerance at -5 °C can be attributed to the inhibition of ice-crystal growth by the IBPs. Significantly, the survival rate of C. elegans at -5 °C was improved by expression of wild-type AnpIBP and maximized by that of TisIBP8, whereas it was lowered when a defective AnpIBP mutant was expressed. These results suggest that the ice-binding ability of IBP has a good correlation with the survival rate of C. elegans during freezing.