062 Antifreeze proteins from Japanese organisms: Functional analyses for their general use

Abstract

Antifreeze protein (AFP) binds to the surface of an ice crystal to inhibit its growth below 0 °C, and also to the membrane surface of a cell to improve its viability above 0 °C. To clarify the mechanism of these two extraordinary functions and to realize a new AFP-utilizing technique, we have been performed exploration of AFPs from Japanese organisms, development of a new mass-preparation method of fish AFP utilizing their muscle homogenate, and structure-function analyses of the AFPs by employing various techniques including X-ray and NMR. The following three topics are presented in this symposium. (1) Mass-purification of fish AFP: In Cryo2009 we reported a simple method to purify an industrial amount (∼kg) of crude AFPI ∼ III and AFGP8 from fish muscle homogenates. In this symposium, we show a development of this method to prepare AFPI of 90% purity from the crude sample, whose production efficiency was 5 g/week in our lab. The biochemical characterization of this purified AFPI will also be presented. (2) Structural determination of fungal AFP: We solved an irregularly ordered solenoid structure of a 223-residue fungal AFP having a triangular cross-section, and determined which of its surfaces forms an ice-binding site (IBS). This IBS is capable of binding to both prism and basal planes, although it has irregularly arrayed surface-bound waters differently to the known hyperactive AFPs. (4) Prolonging cell life-time at 4 °C with AFP: AFP-induced effect on the survival rate was examined for human hepatoma cell (HepG2), rat insulinoma cell (RIN-5F), and bovine blastcyst under hypothermic (+4 °C) temperature. A dramatic increase of the rate was observed at the AFP concentration of 10 mg/ml. The hatching rate of bovine blastcyst after 10-day preservation was improved from 3% to 20% by addition of a recombinant protein of AFPIII. The pregnancy was verified for two recipient heifers so far, for each the 10-day preserved blastcyst with AFP was transferred. The present obtained results will help understanding of the AFP functions and will provide an example of AFP-utilizing technique. Source of Funding: This work was supported by a Grant-in-Aid for scientific research from the Japan Society for the Promotion of Science (JSPS) (No. 23310171) and from Japan Bio-oriented Technology Research Advancement Institution (BRAIN). Conflict of Interest: None declared. s.tsuda@aist.go.jp