Antifreeze Protein Research Team

Antifreeze protein (AFP) initially isolated from blood serum of polar fish in 1960's is an extraordinary bio-molecule that specifically binds to the surface of an ice crystal to inhibit its growth below 0 ℃, and also to the membrane surface of a cell to improve its viability above 0 ℃. We discovered for the first time that approximately 50 kinds of Japanese edible fish also contain AFP, and developed an easy method to obtain ~kg order of natural AFPs from the fish muscle. We also found that an AFP isoform having no antifreeze activity exhibits an extreme cell-preservation ability. Based on the knowledge of biochemical/biophysical properties and structure-function relationships of our original AFPs, the AFP-team is trying to develop new AFP-technologies applicable from industry to medical fields.

In 1990, Prof. Rubinsky and colleagues in the University of California found that AFPs derived from polar fish serum showed a cell protection function in the non-freezing temperature. Thereafter, scientists have been tried to perform the hypothermic storage of germ cells, oocytes, and organs with fish AFPs at the temperature just before freezing (+4 °C). However, because of the scarcity of fish AFP that had to be purified from blood sera of polar fishes, such storage technique was not established well. Here we immersed an insulin production cells (pancreatic islet cell) in a storage solution containing fish AFP, and examined how the survival rate of the cell changed with preservation time at 4 °C. Significantly, it was shown that approximately 60% of pancreatic islet cells could alive even after 120 hrs of preservation period ( PLoS ONE 8(9), 2013, e73643.). The preserved pancreatic islet cells held the insulin secretion ability. It appeared that the cell stock solution showing the best performance includes type I AFP derived from the flatfish, for which a helical structure was assumed (Fig. a). On the basis of a photomicroscope observation that showed a preferable adsorption of AFPs to the lipid bilayer of the pancreatic islet cells (Fig. b), it was suggested that such a membrane-protection ability of AFPs may prolong the life-time of insulinoma cells during the hypothermic exposure.

AFP has been identified from fishes, insects, plants, bacteria, and fungi in the last 4 decades. We solved the first crystal structure of a fungal AFP, and determined which of its surfaces binds to ice (Proc. Natl. Acad. Sci., 2012, 109 (24), 9360-9365.). Our traget was a 223-residue AFP from snow mold fungus,Typhura ishikariensis (photos a and b), which was discovered in 1930s from our place, "a plane of Ishikari" in Hokkaido, Japan. My collaborator (Hoshino, T) characterized the primary sequence of this AFP and named it TisAFP in 2003 (Can. J. Botany, 2003, 81 (12), 1175-1181.). It appeared that TisAFP forms a semipear-shaped overall structure as shown in (c), the right side of which is the putative ice-binding site (IBS). The most striking feature of TisAFP was that it can bind to both prism and basal planes of ice crystal similarly to insect AFP, while TisAFP had irregularly arrayed surface-bound waters (d) differently from the insect AFP (f). These waters were thought to share the positions of ice lattice at the moment AFP attaches onto ice. The following (f) and (g) compare the backbone architecture with illustration between insect AFP and TisAFP. The beta-loops are regularly formed from N to C-termini in insect AFP (f). In contrast, the terminal loops are side by side within an irregularly formed parallel beta-helix of TisAFP as illustrated by the spectrum colors showing blue next to red (g). Additionally, the six helical loops of TisAFP are of different lengths. This unique architecture of TisAFP will locate the surface-bound waters irregularly. The structural features of TisAFP may presumably be adopted in the other fungal AFPs.

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Bioproduction Research Institute

AIST Hokkaido
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