Search Results (17)

Glycopolypeptoids were synthesized and showed effective antifreeze activity, demonstrating their potential as novel antifreeze agents for cryopreservation. These polypeptide analogs offer improved stability and tunability compared with natural antifreeze glycoproteins (AFGPs) and existing synthetic mimics. Using the ring-opening polymerization of N-substituted N-carboxyanhydride monomers followed by click chemistry, glycopolypeptoids with controlled polymerization degrees and varied structures were designed and prepared. Their antifreeze performance was assessed via nanoliter osmometry and ice recrystallization inhibition assays, revealing a strong correlation between the molecular structure and antifreeze activity. The findings highlight glycopolypeptoids as a promising, cost-effective alternative to natural AFGPs, providing new insights into the development of biomimetic cryoprotectants. This study expands the understanding of synthetic antifreeze materials and offers a practical approach for improving cryopreservation efficiency in biomedical and industrial applications. Full article

Understanding characteristic post-source decay (PSD) fragmentation patterns in tandem mass spectrometry (MS/MS) is important for the identification of target molecules. In this study, we explored the characteristic PSD patterns associated with O-linked glycopeptides and their cyclization using the MALDI-TOF/TOF MS analysis of linear and cyclic antifreeze glycoproteins. We performed a comparative analysis of the proton and sodium adduct ions of the peptide backbones of antifreeze glycoproteins, which have a simple repeating sequence, shedding light on the characteristics of the fragmentation of the threonine side chain and that of its cyclized form. Furthermore, the presence or absence of a glycan on the threonine side chain and its substitution with serine caused changes in its fragmentation. These findings are expected to contribute to the prediction of three-dimensional peptide structures and the search for physiologically active O-linked glycopeptides and cyclic (glyco)peptides. Full article

As the most potent ice recrystallization inhibitors, antifreeze glycoproteins (AFGPs) have been extensively studied since their discovery. However, the molecular mechanism of how they inhibit ice growth remains controversial—notably, which group directly contributes to the binding of AFGPs to ice is hotly debated. Here, we use molecular dynamics simulations to investigate the atomistic details of the binding of AFGP8 to ice. We show that the binding of AFGP8 to ice can be divided into three cases: backbone dominant binding (BDB), disaccharide dominant binding (DDB) and weak binding (WB). Hydrogen-bonding and hydrophobic groups contribute equally to the binding of AFGP8 to ice and synergistically promote the binding. The –CH₃ groups promote the contacting of AFGP8 to ice via hydrophobic effect, and the hydrogen-bonding groups anchor AFGP8 to ice surfaces through direct hydrogen bonding with ice. Specially, we verify that the -CONH- groups anchor the backbone of AFGP8 to ice by forming hydrogen bonds with ice surfaces while the –OH groups not only anchor the disaccharide to ice but also slow down the dynamics of the surrounding water. In addition, we reveal that both the backbone and the disaccharide can bind to ice surfaces while the latter is more flexible, which also perturbs the hydrogen bond network of potential ice-like water molecules by swaying in the solution to further enhance its antifreeze activity. This work provides the atomistic details of the ice growth inhibition mechanism of AFGP8, which is helpful for the design of high-efficacy cryoprotectants. Full article

Various alternative compounds have been investigated to prevent icing, one of which includes poly(vinyl) alcohol (PVA), which has shown promising anti-freeze effects. However, determining the optimal structures and formulations of PVA for anti-icing applications has remained a challenge. Building upon our previous work, which used molecular dynamics simulations to assess the effects of hydroxyl group separation distance on ice nucleation, in this work, PVA was modified based upon the structures of antifreeze glycoproteins (AFGPs) found in Antarctic fish, and examined as a potential antifreeze compound. Four different PVA samples with different degrees of hydrolysis were fabricated and subsequently examined for their effects on ice crystallization. The results showed that the modified PVA samples with degrees of hydrolysis of 76% and 66% had an effect on ice crystallization, delaying ice crystallization by an average of approximately 20 min, and even preventing ice crystallization altogether in a small portion of the sample. Meanwhile, other samples with degrees of hydrolysis of 100% and 34% either showed no effect on ice crystallization, shortened the ice crystallization time, and appeared to promote ice nucleation. Full article

Freezing has been widely used for long-term food preservation. However, freezing-thawing (FT) treatment usually influences the texture and structure of food gels such as konjac. For their texture control after FT treatment, it is important to clarify the structural change of food gels during the FT process. In this study, we investigated the aggregated structures of konjac glucomannan (GM) gels during the FT process using simultaneous synchrotron small-angle X-ray/wide-angle X-ray scattering (SAXS/WAXS) techniques. The FT treatment resulted in more crystallization of GM, and consequently, a large increase in compressive stress. In-situ SAXS/WAXS measurements revealed the following findings: on freezing, water molecules came out of the aggregated phase of GM and after the thawing, they came back into the aggregated phase, but the aggregated structure did not return to the one before the freezing; the gel network enhanced the inhomogeneity due to the growth of ice crystals during freezing. Furthermore, we examined the influence of additives such as polyvinyl (alcohol) (PVA) and antifreeze glycoprotein (AFGP) on the mechanical and structural properties of freeze-thawed GM gels. Although the addition of PVA and AFGP suppressed the crystallization of GM, it could not prevent the growth of ice crystals and the increase in the inhomogeneity of the gel network. As a result, the compressive stresses for freeze-thawed GM gels containing PVA or AFGP were significantly higher compared with those of GM gels without FT treatments, although they were lower than those of freeze-thawed GM gels. The findings of this study may be useful for not only the texture control of freeze-thawed foods but also the improvement of the mechanical performance of the biomaterials. Full article

Antifreeze proteins (AFPs) or thermal hysteresis (TH) proteins are biomolecular gifts of nature to sustain life in extremely cold environments. This family of peptides, glycopeptides and proteins produced by diverse organisms including bacteria, yeast, insects and fish act by non-colligatively depressing the freezing temperature of the water below its melting point in a process termed thermal hysteresis which is then responsible for ice crystal equilibrium and inhibition of ice recrystallisation; the major cause of cell dehydration, membrane rupture and subsequent cryodamage. Scientists on the other hand have been exploring various substances as cryoprotectants. Some of the cryoprotectants in use include trehalose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), sucrose, propylene glycol (PG) and glycerol but their extensive application is limited mostly by toxicity, thus fueling the quest for better cryoprotectants. Hence, extracting or synthesizing antifreeze protein and testing their cryoprotective activity has become a popular topic among researchers. Research concerning AFPs encompasses lots of effort ranging from understanding their sources and mechanism of action, extraction and purification/synthesis to structural elucidation with the aim of achieving better outcomes in cryopreservation. This review explores the potential clinical application of AFPs in the cryopreservation of different cells, tissues and organs. Here, we discuss novel approaches, identify research gaps and propose future research directions in the application of AFPs based on recent studies with the aim of achieving successful clinical and commercial use of AFPs in the future. Full article

The de novo birth of functional genes from non-coding DNA as an important contributor to new gene formation is increasingly supported by evidence from diverse eukaryotic lineages. However, many uncertainties remain, including how the incipient de novo genes would continue to evolve and the molecular mechanisms underlying their evolutionary trajectory. Here we address these questions by investigating evolutionary history of the de novo antifreeze glycoprotein (AFGP) gene and gene family in gadid (codfish) lineages. We examined AFGP phenotype on a phylogenetic framework encompassing a broad sampling of gadids from freezing and non-freezing habitats. In three select species representing different AFGP-bearing clades, we analyzed all AFGP gene family members and the broader scale AFGP genomic regions in detail. Codon usage analyses suggest that motif duplication produced the intragenic AFGP tripeptide coding repeats, and rapid sequence divergence post-duplication stabilized the recombination-prone long repetitive coding region. Genomic loci analyses support AFGP originated once from a single ancestral genomic origin, and shed light on how the de novo gene proliferated into a gene family. Results also show the processes of gene duplication and gene loss are distinctive in separate clades, and both genotype and phenotype are commensurate with differential local selective pressures. Full article

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. Full article

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. Full article

Pathogens can manipulate the phenotypic traits of their hosts and vectors, maximizing their own fitness. Among the phenotypic traits that can be modified, manipulating vector behavior represents one of the most fascinating facets. How pathogens infection affects behavioral traits of key insect vectors has been extensively investigated. Major examples include Plasmodium, Leishmania and Trypanosoma spp. manipulating the behavior of mosquitoes, sand flies and kissing bugs, respectively. However, research on how pathogens can modify tick behavior is patchy. This review focuses on current knowledge about the behavioral changes triggered by Anaplasma, Borrelia, Babesia, Bartonella, Rickettsia and tick-borne encephalitis virus (TBEV) infection in tick vectors, analyzing their potential adaptive significance. As a general trend, being infected by Borrelia and TBEV boosts tick mobility (both questing and walking activity). Borrelia and Anaplasma infection magnifies Ixodes desiccation resistance, triggering physiological changes (Borrelia: higher fat reserves; Anaplasma: synthesis of heat shock proteins). Anaplasma infection also improves cold resistance in infected ticks through synthesis of an antifreeze glycoprotein. Being infected by Anaplasma, Borrelia and Babesia leads to increased tick survival. Borrelia, Babesia and Bartonella infection facilitates blood engorgement. In the last section, current challenges for future studies are outlined. Full article

Ixodes scapularis ticks harbor microbial communities including pathogenic and non-pathogenic microbes. Pathogen infection increases the expression of several tick gut proteins, which disturb the tick gut microbiota and impact bacterial biofilm formation. Anaplasma phagocytophilum induces ticks to express I. scapularis antifreeze glycoprotein (IAFGP), a protein with antimicrobial activity, while Borrelia burgdorferi induces the expression of PIXR. Here, we tested the resistance of I. scapularis microbiome to A. phagocytophilum infection, antimicrobial peptide IAFGP, and anti-tick immunity specific to PIXR. We demonstrate that A. phagocytophilum infection and IAFGP affect the taxonomic composition and taxa co-occurrence networks, but had limited impact on the functional traits of tick microbiome. In contrast, anti-tick immunity disturbed the taxonomic composition and the functional profile of tick microbiome, by increasing both the taxonomic and pathways diversity. Mechanistically, we show that anti-tick immunity increases the representation and importance of the polysaccharide biosynthesis pathways involved in biofilm formation, while these pathways are under-represented in the microbiome of ticks infected by A. phagocytophilum or exposed to IAFGP. These analyses revealed that tick microbiota is highly sensitive to anti-tick immunity, while it is less sensitive to pathogen infection and antimicrobial peptides. Results suggest that biofilm formation may be a defensive response of tick microbiome to anti-tick immunity. Full article

The concentration of a protein is highly related to its biochemical properties, and is a key determinant for its biotechnological applications. Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are structurally diverse macromolecules that are capable of binding to embryonic ice crystals below 0 °C, making them useful as protectants of ice-block formation. In this study, we examined the maximal solubility of native AFP I–III and AFGP with distilled water, and evaluated concentration dependence of their ice-binding property. Approximately 400 mg/mL (AFP I), 200 mg/mL (AFP II), 100 mg/mL (AFP III), and >1800 mg/mL (AFGP) of the maximal solubility were estimated, and among them AFGP’s solubility is much higher compared with that of ordinary proteins, such as serum albumin (~500 mg/mL). The samples also exhibited unexpectedly high thermal hysteresis values (2–3 °C) at 50–200 mg/mL. Furthermore, the analysis of fluorescence-based ice plane affinity showed that AFP II binds to multiple ice planes in a concentration-dependent manner, for which an oligomerization mechanism was hypothesized. The difference of concentration dependence between AFPs and AFGPs may provide a new clue to help us understand the ice-binding function of these proteins. Full article

Necessitated by the subzero temperatures and seasonal exposure to ice, various organisms have developed a remarkably effective means to survive the harsh climate of their natural habitats. Their ice-binding (glyco)proteins keep the nucleation and growth of ice crystals in check by recognizing and binding to specific ice crystal faces, which arrests further ice growth and inhibits ice recrystallization (IRI). Inspired by the success of this adaptive strategy, various approaches have been proposed over the past decades to engineer materials that harness these cryoprotective features. In this review we discuss the prospects and challenges associated with these advances focusing in particular on peptidic antifreeze materials both identical and akin to natural ice-binding proteins (IBPs). We address the latest advances in their design, synthesis, characterization and application in preservation of biologics and foods. Particular attention is devoted to insights in structure-activity relations culminating in the synthesis of de novo peptide analogues. These are sequences that resemble but are not identical to naturally occurring IBPs. We also draw attention to impactful developments in solid-phase peptide synthesis and ‘greener’ synthesis routes, which may aid to overcome one of the major bottlenecks in the translation of this technology: unavailability of large quantities of low-cost antifreeze materials with excellent IRI activity at (sub)micromolar concentrations. Full article

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 AFGP_1–5 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 (k_d) of AFGP_1–5 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

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