Stoddard, S.T.; Jin, Y.; Cheng, C.H.*; DeVries, A.L.: Antifreeze Potentiating Protein in Antarctic Notothenioid Fishes

The ability of Antarctic notothenioid fishes to survive in ice-laden waters at -1.9^oC has been attributed to a group of antifreeze glycoproteins (AFGPs). In the absence of AFGPs, the freezing point of body fluids in the fish would be approximately -1^oC due to colligative properties. By binding to ice crystals and inhibiting their growth, AFGPs suppress the freezing point another 1.2^oC. Together, this gives an organismal freezing point of about -2.2^oC, sufficient for the fish to avoid freezing. However, it has also been observed that the freezing point of native notothenioid fish serum can reach as low as -2.7^oC, suggesting that another factor is active in preventing ice growth. We have now discovered a second ice-active serum protein. Alone, this protein displays weak antifreeze activity and does not contribute the remainder of the observed activity in native serum. However, when mixed with AFGPs 1-5, it was found to potentiate antifreeze activity up to 5 or 6 fold, thus accounting for the full freezing point depression observed in native serum. This protein, called antifreeze potentiating protein (AFPP), has a molecular weight of 15,489 kD. It is present at variable concentrations (1 – 4 mg/ml) in the blood of notothenioids; the highest value found in species associated with the iciest habitats. Proteolytic digestion and protein sequence analysis has yielded partial sequences that appear to contain a repeated Ser-Ala-Gly motif. Current work is focusing on the use of primers derived from the protein sequence to generate full length AFPP cDNA by RT-PCR. There are no current sequences in the data similar to the AFPP partial sequence. Therefore AFPP is a novel ice-binding protein that together with AFGPs forms the first example of a two-protein antifreeze system in fish.