Antifreeze protein

• Type I AFP is found in
  shorthorn sculpin. It is the best documented AFP because it was the first to have its three-dimensional structure determined.^[9] Type I AFP consists of a single, long, amphipathic alpha helix, about 3.3-4.5 kD in size. There are three faces to the 3D structure: the hydrophobic, hydrophilic, and Thr-Asx face.^[9]
  • Type I-hyp AFP (where hyp stands for hyperactive) are found in several righteye flounders. It is approximately 32 kD (two 17 kD dimeric molecules). The protein was isolated from the blood plasma of winter flounder. It is considerably better at depressing freezing temperature than most fish AFPs.[10] The ability is partially derived from its many repeats of the Type I ice-binding site.^[11]
• Type II AFPs (e.g. P05140) are found in
  disulfide bonds.^[12] Type II AFPs likely evolved from calcium dependent (c-type) lectins.^[13] Sea ravens, smelt, and herring are quite divergent lineages of teleost. If the AFP gene were present in the most recent common ancestor of these lineages, it is peculiar that the gene is scattered throughout those lineages, present in some orders and absent in others. It has been suggested that lateral gene transfer could be attributed to this discrepancy, such that the smelt acquired the type II AFP gene from the herring.^[14]
• Type III AFPs are found in Antarctic eelpout. They exhibit similar overall hydrophobicity at ice binding surfaces to type I AFPs. They are approximately 6kD in size.^[7] Type III AFPs likely evolved from a sialic acid synthase (SAS) gene present in Antarctic eelpout. Through a gene duplication event, this gene—which has been shown to exhibit some ice-binding activity of its own—evolved into an effective AFP gene by loss of the N-terminal part.^[15]
• Type IV AFPs (P80961) are found in longhorn sculpins. They are alpha helical proteins rich in glutamate and glutamine.
  pyroglutamate residue, a cyclized glutamine residue at its N-terminus.^[16]

The classification of AFPs became more complicated when antifreeze proteins from plants were discovered.^[17] Plant AFPs are rather different from the other AFPs in the following aspects:

1. They have much weaker thermal hysteresis activity when compared to other AFPs.^[18]
2. Their physiological function is likely in inhibiting the recrystallization of ice rather than in preventing ice formation.^[18]
3. Most of them are evolved pathogenesis-related proteins, sometimes retaining antifungal properties.^[18]

Insect AFPs

There are a number of AFPs found in insects, including those from Dendroides, Tenebrio and Rhagium beetles, spruce budworm and pale beauty moths, and midges (same order as flies). Insect AFPs share certain similarities, with most having higher activity (i.e. greater thermal hysteresis value, termed hyperactive) and a repetitive structure with a flat ice-binding surface. Those from the closely related Tenebrio and Dendroides beetles are homologous and each 12–13 amino-acid repeat is stabilized by an internal disulfide bond. Isoforms have between 6 and 10 of these repeats that form a coil, or beta-solenoid. One side of the solenoid has a flat ice-binding surface that consists of a double row of threonine residues.^[6][19] Other beetles (genus Rhagium) have longer repeats without internal disulfide bonds that form a compressed beta-solenoid (beta sandwich) with four rows of threonine residus,^[20] and this AFP is structurally similar to that modelled for the non-homologous AFP from the pale beauty moth.^[21] In contrast, the AFP from the spruce budworm moth is a solenoid that superficially resembles the Tenebrio protein, with a similar ice-binding surface, but it has a triangular cross-section, with longer repeats that lack the internal disulfide bonds. The AFP from midges is structurally similar to those from Tenebrio and Dendroides, but the disulfide-braced beta-solenoid is formed from shorter 10 amino-acids repeats, and instead of threonine, the ice-binding surface consists of a single row of tyrosine residues.^[22] Springtails (Collembola) are not insects, but like insects, they are arthropods with six legs. A species found in Canada, which is often called a "snow flea", produces hyperactive AFPs.^[23] Although they are also repetitive and have a flat ice-binding surface, the similarity ends there. Around 50% of the residues are glycine (Gly), with repeats of Gly-Gly- X or Gly-X-X, where X is any amino acid. Each 3-amino-acid repeat forms one turn of a polyproline type II helix. The helices then fold together, to form a bundle that is two helices thick, with an ice-binding face dominated by small hydrophobic residues like alanine, rather than threonine.^[24] Other insects, such as an Alaskan beetle, produce hyperactive antifreezes that are even less similar, as they are polymers of sugars (xylomannan) rather than polymers of amino acids (proteins).^[25] Taken together, this suggests that most of the AFPs and antifreezes arose after the lineages that gave rise to these various insects diverged. The similarities they do share are the result of convergent evolution.

Sea ice organism AFPs

Many microorganisms living in

Several structures for sea ice AFPs have been solved. This family of proteins fold into a beta helix that form a flat ice-binding surface.^[33] Unlike the other AFPs, there is not a singular sequence motif for the ice-binding site.^[34]

AFP found from the metagenome of the

This independent development of similar adaptations is referred to as convergent evolution.^[4] Evidence for convergent evolution in Northern cod (Gadidae) and Notothenioids is supported by the findings of different spacer sequences and different organization of  introns and exons as well as unmatching AFGP tripeptide sequences, which emerged from duplications of short ancestral sequences which were differently permuted (for the same tripeptide) by each group. These groups diverged approximately 7-15 million years ago. Shortly after (5-15 mya), the AFGP gene evolved from an ancestral pancreatic trypsinogen gene in Notothenioids. AFGP and trypsinogen genes split via a sequence divergence - an adaptation which occurred alongside the cooling and eventual freezing of the Antarctic Ocean. The evolution of the AFGP gene in Northern cod occurred more recently (~3.2 mya) and emerged from a noncoding sequence via tandem duplications in a Thr-Ala-Ala unit. Antarctic notothenioid fish and artic cod, Boreogadus saida, are part of two distinct orders and have very similar antifreeze glycoproteins.^[38] Although the two fish orders have similar antifreeze proteins, cod species contain arginine in AFG, while Antarctic notothenioid do not.^[38] The role of arginine as an enhancer has been investigated in Dendroides canadensis antifreeze protein (DAFP-1) by observing the effect of a chemical modification using 1-2 cyclohexanedione.^[39] Previous research has found various enhancers of this bettles' antifreeze protein including a thaumatin-like protein and polycarboxylates.^[40][41] Modifications of DAFP-1 with the arginine specific reagent resulted in the partial and complete loss of thermal hysteresis in DAFP-1, indicating that arginine plays a crucial role in enhancing its ability.^[39] Different enhancer molecules of DAFP-1 have distinct thermal hysteresis activity.^[41] Amornwittawat et al. 2008 found that the number of carboxylate groups in a molecules influence the enhancing ability of DAFP-1.^[41] Optimum activity in TH is correlated with high concentration of enhancer molecules.^[41] Li et al. 1998 investigated the effects of pH and solute on thermal hysteresis in Antifreeze proteins from Dendrioides canadensis.^[42] TH activity of DAFP-4 was not affected by pH unless the there was a low solute concentration (pH 1) in which TH decreased.^[42] The effect of five solutes; succinate, citrate, malate, malonate, and acetate, on TH activity was reported.^[42] Among the five solutes, citrate was shown to have the greatest enhancing effect.^[42]

This is an example of a proto-ORF model, a rare occurrence where new genes pre exist as a formed open reading frame before the existence of the regulatory element needed to activate them.

In fishes, horizontal gene transfer is responsible for the presence of Type II AFP proteins in some groups without a recently shared phylogeny. In Herring and smelt, up to 98% of introns for this gene are shared; the method of transfer is assumed to occur during mating via sperm cells exposed to foreign DNA.^[43] The direction of transfer is known to be from herring to smelt as herring have 8 times the copies of AFP gene as smelt (1) and the segments of the gene in smelt house transposable elements which are otherwise characteristic of and common in herring but not found in other fishes.^[43]

There are two reasons why many types of AFPs are able to carry out the same function despite their diversity:

1. Although ice is uniformly composed of water molecules, it has many different surfaces exposed for binding. Different types of AFPs may interact with different surfaces.
2. Although the five types of AFPs differ in their
   tertiary structure that facilitates the same interactions with ice.^[4][44]

Antifreeze glycoprotein activity has been observed across several ray-finned species including eelpouts, sculpins, and cod species.[45]^[46] Fish species that possess the antifreeze glycoprotein express different levels of protein activity.^[47] Polar cod (Boreogadus saida) exhibit similar protein activity and properties to the Antarctic species, T. borchgrevinki.^[47] Both species have higher protein activity than saffron cod (Eleginus gracilis).^[47] Ice antifreeze proteins have been reported in diatom species to help decrease the freezing point of organism's proteins.^[26] Bayer-Giraldi et al. 2010 found 30 species from distinct taxa with homologues of ice antifreeze proteins.^[26] The diversity is consistent with previous research that has observed the presence of these genes in crustaceans, insects, bacteria, and fungi.^[8][48][49] Horizontal gene transfer is responsible for the presence of ice antifreeze proteins in two sea diatom species, F. cylindrus and F. curta.^[26]

Mechanisms of action

AFPs are thought to inhibit ice growth by an

Normally, ice crystals grown in solution only exhibit the basal (0001) and prism faces (1010), and appear as round and flat discs.[5] However, it appears the presence of AFPs exposes other faces. It now appears the ice surface 2021 is the preferred binding surface, at least for AFP type I.^[53] Through studies on type I AFP, ice and AFP were initially thought to interact through hydrogen bonding (Raymond and DeVries, 1977). However, when parts of the protein thought to facilitate this hydrogen bonding were mutated, the hypothesized decrease in antifreeze activity was not observed. Recent data suggest hydrophobic interactions could be the main contributor.^[54] It is difficult to discern the exact mechanism of binding because of the complex water-ice interface. Currently, attempts to uncover the precise mechanism are being made through use of molecular modelling programs (molecular dynamics or the Monte Carlo method).^[3][5]

Binding mechanism and antifreeze function

According to the structure and function study on the antifreeze protein from

residues to the oxygens along the ${\displaystyle [01{\overline {1}}2]}$ direction in ice lattice, subsequently stopping or retarding the growth of ice pyramidal planes so as to depress the freeze point.[55]

The above mechanism can be used to elucidate the structure-function relationship of other antifreeze proteins with the following two common features:

1. recurrence of a Thr residue (or any other polar amino acid residue whose side-chain can form a hydrogen bond with water) in an 11-amino-acid period along the sequence concerned, and
2. a high percentage of an Ala residue component therein.^[55]

History

In the 1950s, Norwegian scientist Scholander set out to explain how Arctic fish can survive in water colder than the freezing point of their blood. His experiments led him to believe there was “antifreeze” in the blood of Arctic fish.

angiosperms, including ones eaten by humans.^[59]

They reported their presence in fungi and bacteria as well.

Name change

Recent attempts have been made to relabel antifreeze proteins as ice structuring proteins to more accurately represent their function and to dispose of any assumed negative relation between AFPs and automotive antifreeze, ethylene glycol. These two things are completely separate entities, and show loose similarity only in their function.^[60]

Commercial and medical applications

Numerous fields would be able to benefit from the protection of tissue damage by freezing. Businesses are currently investigating the use of these proteins in:^{[citation needed]}

• Increasing freeze tolerance of crop plants and extending the harvest season in cooler climates
• Improving farm fish production in cooler climates
• Lengthening shelf life of frozen foods
• Improving cryosurgery
• Enhancing preservation of tissues for transplant or transfusion in medicine^[23]
• Therapy for hypothermia
• Human Cryopreservation (Cryonics)

genetically modified yeast to produce antifreeze proteins from fish for use in ice cream production.^[61][62]

They are labeled "ISP" or ice structuring protein on the label, instead of AFP or antifreeze protein.

Recent news

One recent, successful business endeavor has been the introduction of AFPs into ice cream and yogurt products. This ingredient, labelled ice-structuring protein, has been approved by the Food and Drug Administration. The proteins are isolated from fish and replicated, on a larger scale, in genetically modified yeast.^[63]

There is concern from organizations opposed to

allergenic effects in humans.^[7]

As well, the

transgenic process of ice structuring proteins production is widely used in society. Insulin and rennet

are produced using this technology. The process does not impact the product; it merely makes production more efficient and prevents the death of fish that would otherwise be killed to extract the protein.

Currently, Unilever incorporates AFPs into some of its American products, including some Popsicle ice pops and a new line of Breyers Light Double Churned ice cream bars. In ice cream, AFPs allow the production of very creamy, dense, reduced fat ice cream with fewer additives.^[65] They control ice crystal growth brought on by thawing on the loading dock or kitchen table, which reduces texture quality.^[66]

In November 2009, the

Proceedings of the National Academy of Sciences published the discovery of a molecule in an Alaskan beetle that behaves like AFPs, but is composed of saccharides and fatty acids.^[25]

A 2010 study demonstrated the stability of superheated water ice crystals in an AFP solution, showing that while the proteins can inhibit freezing, they can also inhibit melting.[67] In 2021, EPFL and Warwick scientists have found an artificial imitation of antifreeze proteins.^[68]

References

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Further reading

• Haymet AD, Ward LG, Harding MM (1999). "Winter Flounder 'anti-freeze' proteins: Synthesis and ice growth inhibition of analogues that probe the relative importance of hydrophobic and hydrogen bonding interactions". Journal of the American Chemical Society. 121 (5): 941–948.
  ISSN 0002-7863
  .
• Sicheri F, Yang DS (June 1995). "Ice-binding structure and mechanism of an antifreeze protein from winter flounder". Nature. 375 (6530): 427–31.
  S2CID 758990
  .

External links

• Cold, Hard Fact: Fish Antifreeze Produced in Pancreas
• Antifreeze Proteins: Molecule of the Month Archived 2015-11-04 at the Wayback Machine, by David Goodsell, RCSB Protein Data Bank
• Overview of all the structural information available in the PDB for UniProt: Q9GTP0 (Thermal hysteresis or Antifreeze protein) at the PDBe-KB.