Molecular evidence of intertidal habitats selecting for repeated ice-binding protein evolution in invertebrates

ABSTRACTIce-binding proteins (IBPs) have evolved independently in multiple taxonomic groups to improve their survival of sub-zero temperatures. Intertidal invertebrates in temperate and polar regions frequently encounter sub-zero temperatures, yet there is little information on IBPs in these organisms. We hypothesized that there are far more ice-binding proteins than are currently known and that the occurrence of freezing in the intertidal zone selects for these proteins. We compiled a list of genome-sequenced invertebrates across multiple habitats and a list of known IBP sequences and used BLAST to identify a wide array of putative IBPs in those invertebrates. We found that the probability of an invertebrate species having an ice-binding protein was significantly greater in intertidal species as compared to those primarily found in open ocean or freshwater habitats. These intertidal IBPs had high sequence similarity to fish and tick antifreeze glycoproteins and fish type II antifreeze proteins. Previously established classifiers based on machine learning techniques further predicted ice-binding activity in the majority of our newly identified putative IBPs. We investigated the potential evolutionary origin of one putative IBP from the hard-shelled mussel Mytilus coruscus and suggest that it arose through gene duplication and neofunctionalization. We show that IBPs likely readily evolve in response to freezing risk, that there is an array of uncharacterized ice binding proteins and highlight the need for broader laboratory-based surveys of the diversity of ice binding activity across diverse taxonomic and ecological groups.Summary statementIntertidal invertebrates have a disproportionate number of putative ice-binding proteins relative to other habitats. These putative proteins are highly similar to antifreeze glycoproteins and type II antifreeze proteins from fish.

Computational Assessment of Modified Antifreeze Glycoproteins on Ice Nucleation

Antifreeze glycoproteins (AFGPs) found in various fish are used by the organisms to prevent freezing. While these compounds have been studied for their ability to bind to, and prevent the complete crystallization of water, the exact mechanisms by which AFGPs prevent freezing are still undetermined. Therefore, building upon our previous work, this study uses molecular dynamics simulations to assess the effects of hydroxyl group separation distance on AFGP ice nucleation activity. Water droplet crystallization simulations showed that modified AFGP structures containing hydroxyl distances smaller than ~3.0 Å lost their ability to prevent ice crystallization. Furthermore, modified AFGP containing hydroxyl distances of 7.327 Å and 6.160 Å was correlated with a promotion in ice nucleation, as demonstrated by the changes in the energy of the system. This supports the notion that the distance, and therefore, geometry characteristics between the hydroxyl groups located on the saccharide structures play a key role in the ice crystallization inhibition properties of AFGP compounds.

Hydroxyl Group Separation Distances in Anti-Freeze Compounds and Their Effects on Ice Nucleation

Since the discovery of biological antifreeze glycoproteins (AFGPs), which can inhibit ice nucleation, there has been considerable interest in understanding their mechanisms and mimicking them in synthetic polymers. In this study, we used molecular dynamics simulations of modified polyvinyl alcohol (PVA) compounds to show that the hydroxyl (OH) group distance is a key factor in whether certain compounds promote or inhibit ice nucleation. A hydroxyl distance smaller than ~2.8 Å but greater than ~7.1 Å in modified PVA (MPVA) compounds was associated with the promotion of ice nucleation, while a hydroxyl group separation distance of approximately ~5.0 Å was correlated with a delay in ice nucleation, owing to changes in the energy of the system. Thus, these results may help explain some of the mechanisms of current known anti-freeze compounds and may have implications for designing new anti-freeze compounds in the future.

Synthesis and conformational preferences of short analogues of antifreeze glycopeptides (AFGP)

Antifreeze glycoproteins are a class of biological agents which enable living at temperatures below the freezing point of the body fluids. Antifreeze glycopeptides usually consist of repeating tripeptide unit (-Ala-Ala-Thr*-), glycosylated at the threonine side chain. However, on the microscopic level, the mechanism of action of these compounds remains unclear. As previous research has shown, antifreeze activity of antifreeze glycopeptides strongly relies on the overall conformation of the molecule as well an on the stereochemistry of amino acid residues. The desired monoglycosylated analogues with acetylated amino termini and the carboxy termini in form of N-methylamide have been synthesized. Conformational nuclear magnetic resonance (NMR) studies of the designed analogues have shown a strong influence of the stereochemistry of amino acid residues on the peptide chain stability, which could be connected to the antifreeze activity of these compounds. A better understanding of the mechanism of action of antifreeze glycopeptides would allow applying these materials, e.g., in food industry and biomedicine.

The Ensemble of Conformations of Antifreeze Glycoproteins (AFGP8): A Study Using Nuclear Magnetic Resonance Spectroscopy

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.

Effect of Antifreeze Glycoproteins on Organoid Survival during and after Hypothermic Storage

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.

Elucidating the role of key structural motifs in antifreeze glycoproteins

Plausible structural alignment of antifreeze glycoprotein (AFGP) relative to the ice lattice highlighting the role of both Ala and GalNAc methyl groups binding to the ice plane.