Special Issue "Antifreeze Protein: New Insight from Different Approaches"

A special issue of Biomolecules (ISSN 2218-273X).

Deadline for manuscript submissions: closed (2 July 2019).

Special Issue Editors

Prof. Dr. Hidemasa Kondo
E-Mail Website
Guest Editor
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, JAPAN.
Interests: structural biology, industrial protein, antifreeze protein, industrial enzyme
Prof. Dr. Sakae Tsuda
E-Mail Website
Guest Editor
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517 JAPAN
Interests: antifreeze proteins (fish AFPI~III, fish AFGP, fungal beta-helical AFPs)

Special Issue Information

Dear Colleagues,

Antifreeze proteins (AFPs) or ice-binding proteins (IBPs) are polypeptides discovered from vertebrates, plants, fungi, and bacteria that survive in cold environments. Increasing evidence has shown that AFPs can bind to a set of waters constructing ice crystals to inhibit their growth and recrystallization at higher subzero temperatures. AFPs are also known to interact with membranes to elongate the life time of various cells near 0°C. Since these functions are related with various research fields and that is worth to be considered intensively, this Special Issue focuses on both basics and technological insights of AFPs. The issue welcomes articles on this unique category and its many different approaches, which may include AFP, IBP, antifreeze glycoprotein (AFGP), ice-binding, ice-plane affinity, thermal hysteresis, crystal growth, ice recrystallization, ice nucleation, AFP-mimetics, tandem repeats, structure, dynamics, water, hydration, molecular dynamics (MD) simulation, chemical synthesis, mass-preparation, evolution, lateral gene transfer, and cell and tissue preservation.

Prof. Dr. Sakae Tsuda
Prof. Dr. Hidemasa Kondo
Guest Editors

Manuscript Submission Information

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Keywords

  • AFP
  • IBP
  • antifreeze glycoprotein (AFGP)
  • ice-binding
  • ice-plane affinity
  • thermal hysteresis
  • crystal growth
  • ice recrystallization
  • ice nucleation
  • AFP-mimetics
  • tandem repeats
  • structure
  • dynamics
  • water
  • hydration
  • molecular dynamics (MD) simulation
  • chemical synthesis
  • mass-preparation
  • evolution
  • lateral gene transfer
  • and cell and tissue preservation

Published Papers (9 papers)

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Research

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Open AccessArticle
Ice Nucleation Properties of Ice-binding Proteins from Snow Fleas
Biomolecules 2019, 9(10), 532; https://doi.org/10.3390/biom9100532 - 25 Sep 2019
Abstract
Ice-binding proteins (IBPs) are found in many organisms, such as fish and hexapods, plants, and bacteria that need to cope with low temperatures. Ice nucleation and thermal hysteresis are two attributes of IBPs. While ice nucleation is promoted by large proteins, known as [...] Read more.
Ice-binding proteins (IBPs) are found in many organisms, such as fish and hexapods, plants, and bacteria that need to cope with low temperatures. Ice nucleation and thermal hysteresis are two attributes of IBPs. While ice nucleation is promoted by large proteins, known as ice nucleating proteins, the smaller IBPs, referred to as antifreeze proteins (AFPs), inhibit the growth of ice crystals by up to several degrees below the melting point, resulting in a thermal hysteresis (TH) gap between melting and ice growth. Recently, we showed that the nucleation capacity of two types of IBPs corresponds to their size, in agreement with classical nucleation theory. Here, we expand this finding to additional IBPs that we isolated from snow fleas (the arthropod Collembola), collected in northern Israel. Chemical analyses using circular dichroism and Fourier-transform infrared spectroscopy data suggest that these IBPs have a similar structure to a previously reported snow flea antifreeze protein. Further experiments reveal that the ice-shell purified proteins have hyperactive antifreeze properties, as determined by nanoliter osmometry, and also exhibit low ice-nucleation activity in accordance with their size. Full article
(This article belongs to the Special Issue Antifreeze Protein: New Insight from Different Approaches)
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Open AccessCommunication
The Impact of Salts on the Ice Recrystallization Inhibition Activity of Antifreeze (Glyco)Proteins
Biomolecules 2019, 9(8), 347; https://doi.org/10.3390/biom9080347 - 06 Aug 2019
Cited by 1
Abstract
Antifreeze (glyco)proteins (AF(G)Ps) have received increasing attention as potential cryopreservation agents since their discovery in the 1970s. While cryopreservation strategies for specific cells (such as red blood cells) are successful and widely implemented, preservation of other cell types, tissues and whole organs remains [...] Read more.
Antifreeze (glyco)proteins (AF(G)Ps) have received increasing attention as potential cryopreservation agents since their discovery in the 1970s. While cryopreservation strategies for specific cells (such as red blood cells) are successful and widely implemented, preservation of other cell types, tissues and whole organs remains challenging. This is due to the multifactorial nature of the freeze-thaw damage, the complexity of preserving biological matter and the (country-to-country) variability of the employed procedures and regulations. AF(G)Ps are well-known for their ability to modulate ice crystal growth morphology and ice recrystallization inhibition (IRI), both of which are considered key contributors to freeze-thaw damage. To date, however, the impact of AF(G)Ps on cell survival remains at best partially understood as conflicting results on the benefits or disadvantages of including AF(G)P in cryopreservation strategies remain unelucidated. We hypothesize that variability in the additives in the cryopreservation media contributes to the observed discrepancies. To critically examine this idea, we monitored the inhibition of ice recrystallization by AF(G)P in the presence of various salts using a quantitative analysis of optical microscopy images via the Lifshitz-Slyozov-Wagner (LSW) theory for Oswald ripening. We found that the addition of salts, which are used in culture and cryopreservation media, enhances the IRI activity of AF(G)Ps, and that the magnitude of the enhancement was in line with the Hofmeister series. The size of ice crystals grown in AFGP1–5 and type III AFP samples containing chloride, phosphate and citrate ions were statistically smaller after 90 min of incubation than crystals grown in the absence of these salts. The ice recrystallization rates (kd) of AFGP1–5 and type III AFP samples prepared at a fixed overall ionic strength of 100 mM progressively decreased following the Hofmeister series for anions. Our results demonstrate that the performance of AF(G)Ps is significantly influenced by additives present in common cryopreservation media. It is thus important to conduct excipient compatibility experiments to identify potential incompatibilities between additives and AF(G)Ps in cryopreservation formulations. Full article
(This article belongs to the Special Issue Antifreeze Protein: New Insight from Different Approaches)
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Open AccessArticle
The Ensemble of Conformations of Antifreeze Glycoproteins (AFGP8): A Study Using Nuclear Magnetic Resonance Spectroscopy
Biomolecules 2019, 9(6), 235; https://doi.org/10.3390/biom9060235 - 17 Jun 2019
Abstract
The primary sequence of antifreeze glycoproteins (AFGPs) is highly degenerate, consisting of multiple repeats of the same tripeptide, Ala–Ala–Thr*, in which Thr* is a glycosylated threonine with the disaccharide beta-d-galactosyl-(1,3)-alpha-N-acetyl-d-galactosamine. AFGPs seem to function as intrinsically disordered proteins, presenting [...] Read more.
The primary sequence of antifreeze glycoproteins (AFGPs) is highly degenerate, consisting of multiple repeats of the same tripeptide, Ala–Ala–Thr*, in which Thr* is a glycosylated threonine with the disaccharide beta-d-galactosyl-(1,3)-alpha-N-acetyl-d-galactosamine. AFGPs seem to function as intrinsically disordered proteins, presenting challenges in determining their native structure. In this work, a different approach was used to elucidate the three-dimensional structure of AFGP8 from the Arctic cod Boreogadus saida and the Antarctic notothenioid Trematomus borchgrevinki. Dimethyl sulfoxide (DMSO), a non-native solvent, was used to make AFGP8 less dynamic in solution. Interestingly, DMSO induced a non-native structure, which could be determined via nuclear magnetic resonance (NMR) spectroscopy. The overall three-dimensional structures of the two AFGP8s from two different natural sources were different from a random coil ensemble, but their “compactness” was very similar, as deduced from NMR measurements. In addition to their similar compactness, the conserved motifs, Ala–Thr*–Pro–Ala and Ala–Thr*–Ala–Ala, present in both AFGP8s, seemed to have very similar three-dimensional structures, leading to a refined definition of local structural motifs. These local structural motifs allowed AFGPs to be considered functioning as effectors, making a transition from disordered to ordered upon binding to the ice surface. In addition, AFGPs could act as dynamic linkers, whereby a short segment folds into a structural motif, while the rest of the AFGPs could still be disordered, thus simultaneously interacting with bulk water molecules and the ice surface, preventing ice crystal growth. Full article
(This article belongs to the Special Issue Antifreeze Protein: New Insight from Different Approaches)
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Open AccessFeature PaperArticle
Laboratory-Scale Isolation of Insect Antifreeze Protein for Cryobiology
Biomolecules 2019, 9(5), 180; https://doi.org/10.3390/biom9050180 - 09 May 2019
Abstract
Micromolar concentrations of hyperactive antifreeze proteins (AFPs) from insects can prevent aqueous solutions from freezing down to at least −6 °C. To explore cryopreservation of cells, tissues and organs at these temperatures without ice formation, we have developed a protocol to reliably produce [...] Read more.
Micromolar concentrations of hyperactive antifreeze proteins (AFPs) from insects can prevent aqueous solutions from freezing down to at least −6 °C. To explore cryopreservation of cells, tissues and organs at these temperatures without ice formation, we have developed a protocol to reliably produce ultrapure Tenebrio molitor AFP from cold-acclimated beetle larvae reared in the laboratory. The AFP was prepared from crude larval homogenates through five cycles of rotary ice-affinity purification, which can be completed in one day. Recovery of the AFP at each step was >90% and no impurities were detected in the final product. The AFP is a mixture of isoforms that are more active in combination than any one single component. Toxicity testing of the purified AFP in cell culture showed no inhibition of cell growth. The production process can easily be scaled up to industrial levels, and the AFP used in cryobiology applications was recovered for reuse in good yield and with full activity. Full article
(This article belongs to the Special Issue Antifreeze Protein: New Insight from Different Approaches)
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Open AccessArticle
Calcium-Binding Generates the Semi-Clathrate Waters on a Type II Antifreeze Protein to Adsorb onto an Ice Crystal Surface
Biomolecules 2019, 9(5), 162; https://doi.org/10.3390/biom9050162 - 27 Apr 2019
Abstract
Hydration is crucial for a function and a ligand recognition of a protein. The hydration shell constructed on an antifreeze protein (AFP) contains many organized waters, through which AFP is thought to bind to specific ice crystal planes. For a Ca2+-dependent [...] Read more.
Hydration is crucial for a function and a ligand recognition of a protein. The hydration shell constructed on an antifreeze protein (AFP) contains many organized waters, through which AFP is thought to bind to specific ice crystal planes. For a Ca2+-dependent species of AFP, however, it has not been clarified how 1 mol of Ca2+-binding is related with the hydration and the ice-binding ability. Here we determined the X-ray crystal structure of a Ca2+-dependent AFP (jsAFP) from Japanese smelt, Hypomesus nipponensis, in both Ca2+-bound and -free states. Their overall structures were closely similar (Root mean square deviation (RMSD) of Cα = 0.31 Å), while they exhibited a significant difference around their Ca2+-binding site. Firstly, the side-chains of four of the five Ca2+-binding residues (Q92, D94 E99, D113, and D114) were oriented to be suitable for ice binding only in the Ca2+-bound state. Second, a Ca2+-binding loop consisting of a segment D94–E99 becomes less flexible by the Ca2+-binding. Third, the Ca2+-binding induces a generation of ice-like clathrate waters around the Ca2+-binding site, which show a perfect position-match to the waters constructing the first prism plane of a single ice crystal. These results suggest that generation of ice-like clathrate waters induced by Ca2+-binding enables the ice-binding of this protein. Full article
(This article belongs to the Special Issue Antifreeze Protein: New Insight from Different Approaches)
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Open AccessArticle
Freeze Tolerance in Sculpins (Pisces; Cottoidea) Inhabiting North Pacific and Arctic Oceans: Antifreeze Activity and Gene Sequences of the Antifreeze Protein
Biomolecules 2019, 9(4), 139; https://doi.org/10.3390/biom9040139 - 06 Apr 2019
Abstract
Many marine species inhabiting icy seawater produce antifreeze proteins (AFPs) to prevent their body fluids from freezing. The sculpin species of the superfamily Cottoidea are widely found from the Arctic to southern hemisphere, some of which are known to express AFP. Here we [...] Read more.
Many marine species inhabiting icy seawater produce antifreeze proteins (AFPs) to prevent their body fluids from freezing. The sculpin species of the superfamily Cottoidea are widely found from the Arctic to southern hemisphere, some of which are known to express AFP. Here we clarified DNA sequence encoding type I AFP for 3 species of 2 families (Cottidae and Agonidae) belonging to Cottoidea. We also examined antifreeze activity for 3 families and 32 species of Cottoidea (Cottidae, Agonidae, and Rhamphocottidae). These fishes were collected in 2013–2015 from the Arctic Ocean, Alaska, Japan. We could identify 8 distinct DNA sequences exhibiting a high similarity to those reported for Myoxocephalus species, suggesting that Cottidae and Agonidae share the same DNA sequence encoding type I AFP. Among the 3 families, Rhamphocottidae that experience a warm current did not show antifreeze activity. The species inhabiting the Arctic Ocean and Northern Japan that often covered with ice floe showed high activity, while those inhabiting Alaska, Southern Japan with a warm current showed low/no activity. These results suggest that Cottoidea acquires type I AFP gene before dividing into Cottidae and Agonidae, and have adapted to each location with optimal antifreeze activity level. Full article
(This article belongs to the Special Issue Antifreeze Protein: New Insight from Different Approaches)
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Open AccessArticle
Effect of Antifreeze Glycoproteins on Organoid Survival during and after Hypothermic Storage
Biomolecules 2019, 9(3), 110; https://doi.org/10.3390/biom9030110 - 19 Mar 2019
Cited by 2
Abstract
We study the effect of antifreeze glycoproteins (AFGPs) on the survival of organoids under hypothermic conditions. We find that the survival of organoids in cold conditions depends on their developmental stage. Mature organoids die within 24 h when being stored at 4 °C, [...] Read more.
We study the effect of antifreeze glycoproteins (AFGPs) on the survival of organoids under hypothermic conditions. We find that the survival of organoids in cold conditions depends on their developmental stage. Mature organoids die within 24 h when being stored at 4 °C, while cystic organoids can survive up to 48 h. We find that in the presence of AFGPs, the organoid survival is prolonged up to 72 h, irrespective of their developmental stage. Fluorescence microscopy experiments reveal that the AFGPs predominately localize at the cell surface and cover the cell membranes. Our findings support a mechanism in which the positive effect of AFGPs on cell survival during hypothermic storage involves the direct interaction of AFGPs with the cell membrane. Our research highlights organoids as an attractive multicellular model system for studying the action of AFGPs that bridges the gap between single-cell and whole-organ studies. Full article
(This article belongs to the Special Issue Antifreeze Protein: New Insight from Different Approaches)
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Open AccessArticle
Effects of Winter Flounder Antifreeze Protein on the Growth of Ice Particles in an Ice Slurry Flow in Mini-Channels
Biomolecules 2019, 9(2), 70; https://doi.org/10.3390/biom9020070 - 18 Feb 2019
Cited by 1
Abstract
The control of ice growth in ice slurry is important for many fields, including (a) the cooling of the brain during cardiac arrest, (b) the storage and transportation of fresh fish and fruits, and (c) the development of distributed air-conditioning systems. One of [...] Read more.
The control of ice growth in ice slurry is important for many fields, including (a) the cooling of the brain during cardiac arrest, (b) the storage and transportation of fresh fish and fruits, and (c) the development of distributed air-conditioning systems. One of the promising methods for the control is to use a substance such as antifreeze protein. We have observed and report here growth states of ice particles in both quiescent and flowing aqueous solutions of winter flounder antifreeze proteins in mini-channels with a microscope. We also measured ice growth rates. Our aim was to improve the levels of ice growth inhibition by subjecting the antifreeze protein solution both to preheating and to concentrating by ultrafiltration. We have found that the ice growth inhibition by the antifreeze protein decreased in flowing solutions compared with that in quiescent solutions. In addition, unlike unidirectional freezing experiments, the preheating of the antifreeze protein solution reduced the ice growth inhibition properties. This is because the direction of flow, containing HPLC6 and its aggregates, to the ice particle surfaces can change as the ice particle grows, and thus the probability of interaction between HPLC6 and ice surfaces does not increase. In contrast to this, ultrafiltration after preheating the solution improved the ice growth inhibition. This may be due to the interaction between ice surfaces and many aggregates in the concentrates. Full article
(This article belongs to the Special Issue Antifreeze Protein: New Insight from Different Approaches)
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Review

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Open AccessReview
The Use of Antifreeze Proteins in the Cryopreservation of Gametes and Embryos
Biomolecules 2019, 9(5), 181; https://doi.org/10.3390/biom9050181 - 09 May 2019
Cited by 2
Abstract
The cryopreservation of gametes and embryos is a technique widely used in reproductive biology. This technology helps in the reproductive management of domesticated animals, and it is an important tool for gene banking and for human-assisted reproductive technologies. Antifreeze proteins are naturally present [...] Read more.
The cryopreservation of gametes and embryos is a technique widely used in reproductive biology. This technology helps in the reproductive management of domesticated animals, and it is an important tool for gene banking and for human-assisted reproductive technologies. Antifreeze proteins are naturally present in several organisms exposed to subzero temperatures. The ability for these proteins to inhibit ice recrystallization together with their ability to interact with biological membranes makes them interesting molecules to be used in cryopreservation protocols. This mini-review provides a general overview about the use of antifreeze proteins to improve the short and long term storage of gametes and embryos. Full article
(This article belongs to the Special Issue Antifreeze Protein: New Insight from Different Approaches)
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