Identification and Characterization of Protein Enhancers of Antifreeze Proteins from Overwintering Beetle Larvae Dendroides Canadensis

Larvae of the beetle Dendroides canadensis produce a family of antifreeze proteins (DAFPs), four of which (DAFP-1, -2, -4, and -6) are in the hemolymph. Antifreeze proteins (AFPs) lower the non-colligative freezing point of water (in the presence of ice) below the melting point, producing a difference between the freezing and melting points termed thermal hysteresis activity (THA). This THA is dependent upon AFP specific activity, concentration, and the presence of enhancers. Enhancers may be low molecular mass enhancers such as glycerol, or other proteins. The protein enhancers complex with the DAFPs, thereby blocking a larger surface area of the potential seed ice crystal and/or making the complex harder for ice to overgrow, and consequently lowering the freezing point. A yeast two-hybrid screen was performed using certain hemolymph DAFPs as'bait' to identify endogenous protein enhancers. Among the positive proteins identified as with the bait DAFPs, and confirmed by co-immunoprecipitation, were other DAFPs as well as a thuamatin-like protein. When different DAFPs were combined, those identified by the yeast two-hybrid screen as positives exhibited a synergistic enhancement of THA. In contrast, those DAFPs which the screen indicated did not interact, failed to enhance one anothers. DAFPs -1 and -2 interact and enhance one another. Point mutations of DAFP-2 indicated that both of the two amino acid residues that differ between DAFPs -1 and -2 were required for interaction. The thaumatin-like protein also significantly enhanced the THA of DAFPs-1 and -2. Glycerol enhanced the THA of the DAFPs only when proteins known to interact were present in the test solution. Addition of glycerol to a test solution containing only one DAFP did not produce enhancement. Therefore, glycerol enhances activity by stimulating interactions between DAFPs and between DAFPs and enhancer proteins, such as the thaumatin-like protein. Native DAFP-6 lacks THA, however, mutation of any of the four residues that differ between DAFP-6 and DAFP-4 (which is active) produced mutated DAFP-6 with low THA. RNAi technology was shown to be useful to knock-down dafp transcripts with injection of dsRNA of DAFP-1.