Grounds for winning the 2019 Society Award of
the Japan Society for Cryobiology and Cryotechnology
Functional analysis of antifreeze protein toward its practical applications
Sakae Tsuda
Chief Senior Researcher of National Institute of Advanced Industrial Scienceand Technology (AIST) and Professor of Hokkaido University (HU)

Dr. Sakae Tsuda received his bachelor’s and master’s degrees (1985 and 1987) from
Hokkaido University (HU) in Japan (JPN). He further continued doctor course student ofHU, and quitted it to start working as a teaching profession of high-resolution NMR
laboratory operated in HU. He obtained Ph.D. degree (Doctor of Science) in 1992 from
HU and moved to Prof. Brian Sykes’ lab in U of Alberta (CAN) as a postdoctoral fellow
to work on the NMR structural analysis of a muscle protein troponin-C. He returned toJPN in 1994 as an NMR technical advisor of a company (JEOL), and then invited to AIST
as a senior researcher in 1995. He obtained the AIST President Award in 2000 for
excellence of his work regarding NMR analysis of the structure-function relationship of
low-temperature adapted biomolecules. He joined this society in 2003, and has beenworking as a commissioner from 2009. He organized 2nd International Ice-Binding
Protein Conference (IBP2014) at Sapporo in 2014, and also hosted 62th annual meetingof this society at Sapporo in 2017. He has joined editorial board member of several
international journals including Scientific Reports. He is now a chief senior researcher ofAIST and a professor of HU with 3 staff members and approximately 10 MC & DC
students to work on the structure and functional analysis of antifreeze proteins (AFPs).

Dr. Sakae Tsuda explored antifreeze proteins (AFPs) from Japanese organisms, suchas, fishes, plants, insects, fungi, and bacteria. He has been clarified DNA sequence, aminoacid sequence, biochemical property, ice-binding function, 2ndary and tertiary structure,cell-preservation ability, and structure-function relationship of each AFP species very
carefully. He discovered that numbers of fishes living in Japan, some of which are easily
available in general food markets, contain type I−III AFPs (AFP I−III) and antifreeze
glycoprotein (AFGP). Such discovery first demonstrated that AFPs and AFGP are notspecial proteins for the organisms in polar area, but are widely distributed in animals andplants living in the middle latitudes that cover Asian countries. He also developed a novel
mass-preparation technique of type I−III AFPs and antifreeze glycoprotein (AFGP) byutilizing muscle of Japanese fishes for the first time, and succeeded theircommercialization in 2016 as the consequence of collaboration studies for many years
with a Japanese company. More details are described below.
(1) Structural and functional analysis of AFP identified from Japanese organisms
He demonstrated that primary sequence and functional property of AFP I−III
contained in Japanese fishes are very unique, and are different from those previously
isolated from animals living in north America. For example, the AFP I from Barfin plaiceconsists of an unusually high content of the hydrophobic alanine, though it exhibited an
extremely high water solubility (~600 mg/mL). The AFP II from Longsnout poacher has
antifreeze activity without Ca2+ by a unique ability to expand its target area to whole ice
crystal planes with increasing the concentration. The AFP III from notched-fin eelpout
consists of 13 isoforms categorized into 6 SP-sepharose binding isoforms and 7 QAEsepharose binding isoforms, although the corresponding AFP from ocean pout has only 1
QAE-isoform. He synthesized many activity-modified mutants of this AFP III todetermine their crystal structures, and discovered that the strongest mutant accompanies“ice-like” water network on its ice-binding site. An observation that several waters in thisnetwork showed complementary position match with the waters constructing a single ice
crystal allowed him to hypothesize an ice-binding model of AFP III that utilizes the icelike water network. He determined both NMR and X-ray structures of Japanese original
AFPs to deposit to Protein Data Bank (PDB), which includes first crystal structure of afungal AFP isolated from Typhula ishikariensis. On the basis of such structural biologic
approach, he further clarified the evolutional origin of sculpin AFPs, the existence ofhorizontal gene transfer between bacteria and fungi, and the ability of AFP to bind to lipid
bilayer that prolongs the life time of a cell.
(2) Development of mass-preparation method of AFP for its application
An ice block that we generally see consists of infinitive numbers of tiny single ice
crystals created in water at the moment of freezing. The crystals grow and merge together
to form an ice block, if we keep the temperature below 0°C. Type I–III AFPs and AFGP

accumulate on the surfaces of such embryonic ice crystals to inhibit their growth andmerging, resulting an aggregate formation of the ice crystals instead of an ice block. Dr.
Sakae Tsuda noticed that this AFP function is useful for preservation of a variety of watercontaining materials such as processed foods, soups, ice creams, noodles, breads,vegetables, seeds, drinks, alcohol, medicines, cosmetics, gels, cells, tissues, and organs.
If AFPs can keep the size of each ice crystal to minimum, they may greatly improve theeffectiveness of preservation. In addition, fish-derived AFPs bind to the lipid bilayer toprolong the lifetime of cells under hypothermic condition (+4°C), which should beapplicable to short-term cell preservation or “cell pausing”. Each AFP and AFGP sample
is always a mixture of 2–13 isoforms, which function together far more effectively thanany single isoform. He has therefore been trying to develop mass-preparation method ofquality products of fish type I–III AFPs and AFGP, and examined their applicability inboth industrial and medical fields. He offered these AFP samples to numbers ofuniversities and institutes for free, and made significant contributions to advance bothbasic studies and applications of a variety of researchers. A unique example of his
developed AFP technique is the fabrication of highly porous material using “gelation &freezing method”. Another example is the AFP-containing cell-preservation fluid, which
actually prolonged the life time of a bovine cell for 10 days at 4°C to give birth to ahealthy calf. From now on, the quality products of AFP and AFGP developed by Tsuda
will realize many more advanced techniques.
All of these results show that Dr. Sakae Tsuda made a remarkable contribution to oursociety through his original activities on cryobiology and cryotechnology. Therefore, we
determined that he deserves 2019 Society Award of the Japan Society for Cryobiology
and Cryotechnology.