Antifreeze Protein Type Research Articles

Antifreeze protein dispersion in eelpouts and related fishes reveals migration and climate alteration within the last 20 Ma.

Antifreeze proteins inhibit ice growth and are crucial for the survival of supercooled fish living in icy seawater. Of the four antifreeze protein types found in fishes, the globular type III from eelpouts is the one restricted to a single infraorder (Zoarcales), which is the only clade know to have antifreeze protein-producing species at both poles. Our analysis of over 60 unique antifreeze protein gene sequences from several Zoarcales species indicates this gene family arose around 18 Ma ago, in the Northern Hemisphere, supporting recent data suggesting that the Arctic Seas were ice-laden earlier than originally thought. The Antarctic was subject to widespread glaciation over 30 Ma and the Notothenioid fishes that produce an unrelated antifreeze glycoprotein extensively exploited the adjoining seas. We show that species from one Zoarcales family only encroached on this niche in the last few Ma, entering an environment already dominated by ice-resistant fishes, long after the onset of glaciation. As eelpouts are one of the dominant benthic fish groups of the deep ocean, they likely migrated from the north to Antarctica via the cold depths, losing all but the fully active isoform gene along the way. In contrast, northern species have retained both the fully active (QAE) and partially active (SP) isoforms for at least 15 Ma, which suggests that the combination of isoforms is functionally advantageous.

Addition of antifreeze protein type I or III to extenders for ram sperm cryopreservation

Antifreeze proteins (AFP) play an important role in cellular survival at sub-zero temperatures. This study assessed the effect of AFP type I or III in semen extender (TRIS-egg yolk) for ram sperm cryopreservation. Pooled semen of four rams were allocated into five treatments: Control (CONT, without AFP); AFP Type I [0.1 (AFPI-0.1) or 0.5 (AFPI-0.5) μg/mL]; or III [0.1 (AFPIII-0.1) or 0.5 (AFPIII-0.5) μg/mL], and then frozen in six replicates. Treatments affected kinetic parameters, plasma membrane integrity and morphology (P < 0.05). The AFPIII-0.1 presented lesser total motility. Linearity was greater in AFPI-0.1, AFPI-0.5 and AFPIII-0.5 and straightness was greater in all AFP-supplemented extenders. Plasma membrane integrity was greater in AFPI-0.1 and AFPI-0.5. All AFP groups had greater percentage of normal sperm than CONT. No differences (P > 0.05) were observed in hypoosmotic test, sperm acrosome status, mitochondrial activity, chromatin condensation, perivitelline membrane binding rate and lipoperoxidation. In conclusion, the use of AFP, predominantly type I, may increase sperm cell protection during cryopreservation, with no adverse effect on potential fertilization capacity or increase in reactive oxygen species, being a potential cryoprotectant to ram sperm.

Physiological and gene expression analysis of the Manila clam Ruditapes philippinarum in response to cold acclimation

Overwinter mortality of the Manila clam (Ruditapes philippinarum) is a major impediment to the aquaculture industry in China. Cold tolerance ability has a tremendous impact on the survivability of R. philippinarum during the overwintering season. In this study, we evaluated the effects of acute and chronic cold stress on the expression of Cold Shock Domain-containing E1 (CSDE1) and Antifreeze protein type II (AFPII) genes and the activities of lysozyme (LZM), catalase (CAT), and superoxide dismutase (SOD) in three cultivated strains (zebra, white, and white zebra) and two wild populations (northern and southern) of R. philippinarum. Under acute and chronic cold stress, the expression levels of CSDE1 and AFPII mRNA in the gills and hepatopancreas were significantly increased in all populations, but the increase varied among different strains and populations. Under acute cold stress, SOD activity significantly decreased in the two wild populations and the white zebra strain. LZM activity significantly decreased but CAT activity significantly increased in selected strains and populations after acute low temperature stress (P < 0.05). Under chronic cold stress, SOD activity significantly increased in the northern population and white zebra strain, while CAT activity significantly increased in the southern population and the white and zebra strains. These results provide useful information about the Manila clam response to cold stress that may be applied to improve the low temperature resistance of Manila clams in aquaculture environments.

Efficacy of antifreeze proteins from Clupea harangues and Anarhichas minor on gas hydrate inhibition via cell surface display

The expression of anti-freeze proteins (AFPs) on the Escherichia coli using the cell surface display (CSD) strategy was investigated to determine the feasibility of the method as a potential application of kinetic hydrate inhibitors. Monomers to hexamers of type II and III AFPs were displayed on the surfaces of bacterial cells. The kinetic inhibition performance of the AFPs on the synthetic natural gas hydrate formation was tested. The displayed AFPs exhibited higher KHI effectiveness with the increase in the number of peptides attached to the cells. In addition, best KHI performance of the AFPs could be achieved with higher dosages of the cultures when the concentration of the inducer is optimized, regardless of AFP type. Only a small portion of AFPs in the cell cultures exhibited considerable KHI performance, which implies that the CSD could be an effective strategy, reducing the cost for the production and purification of the AFPs.

Tomato seeds pretreated with Antifreeze protein type I (AFP I) promotes the germination under cold stress by regulating the genes involved in germination process

ABSTRACTThis study was conducted to investigate the involvement of antifreeze proteins (AFPs; type I and III) in the germination mechanism of tomato seeds under low temperature stress. Germination of the seeds grown at a room temperature (25°C) was observed on 5 days after sowing (DAS), while all seeds exposed to a low temperature started to germinate at 16 days after sowing (DAS). However, in comparison with control seeds (0 µg/l), seeds treated with AFP I (100, 300, or 500 µg/l) germinated earlier and at a higher percentage until 20 DAS, and seeds treated with 100 µg/l AFP I showed the highest percentage of germination. Surprisingly, AFP III did not significantly increase germination, and the rate was lower among 500 µg/l AFP III-treated seeds compared with control seeds (0 µg/l). The transcription levels of the plasma membrane-associated H+-ATPase gene and antioxidant-related superoxide dismutase (SOD) and catalase 1 (CAT1) genes were analyzed, and the transcription levels of the genes in the seeds grown at 25°C were relatively low. For low temperature-treated seeds, H+-ATPase in control seeds (0 µg/l) was higher compared with that in AFP I-treated seeds and was lower compared with that in AFP III-treated seeds. The expression levels of the antioxidant-related genes (SOD and CAT1) were lower in AFP I-treated seeds than in control seeds (0 µg/l); however, they were higher in AFP III-treated seeds than in control seeds (0 µg/l). Overall, compared with AFP III, AFP I may potentially function as a cold-protective agent by modulating the genes associated with seed germination.

Seminal plasma transferrin effects on cryopreserved common carp Cyprinus carpio sperm and comparison with bovine serum albumin and antifreeze proteins

In the current study, there was evaluation of the cryopreservation effectiveness of common carp Cyprinus carpio sperm when cryopreservation medium was supplemented with proteins. Semen was diluted with Kurokura’s extender composing 180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl2, 2.38 mM NaHCO3, and 10% dimethylsulfoxide (DMSO). Cryopreservation medium was supplemented with purified seminal plasma transferrin (Tf), bovine serum albumin (BSA) or antifreeze protein (AFP) Types I and III. Concentration of proteins evaluated was 0.1 μg/ml, 1 μg/ml, and 10 μg/ml. Motility and curvilinear velocity of spermatozoa was evaluated by the Computer Assisted Semen Analyzer (CASA). The percent of motile cells and spermatozoa curvilinear velocity of frozen-thawed sperm with supplementation of Tf and AFP III at all concentrations were greater compared to samples with no added proteins. The protective effect of BSA and AFP I was less and dose-dependent. Thus, it is concluded that incorporation of Tf in the extender before freezing improves crypreservation of common carp spermatozoa whereas supplementation with AFP III in greater concentrations was more effective.

Effects of antifreeze proteins on cryopreserved sterlet (Acipenser ruthenus) sperm motility variables and fertilization capacity

The effect of antifreeze proteins on sterlet, Acipenser ruthenus sperm motility variables and fertilization rate were investigated after cryopreservation. Two types of antifreeze proteins (AFPI or AFPIII) were used at concentrations of 0.1, 1, 10 and 100 μg/mL. The motility variables of fresh and cryopreserved sperm with and without addition of antifreeze proteins were evaluated by the Computer Assisted Semen Analyzer (CASA). The fertilization rate using about 200,000 spermatozoa per egg was evaluated after 54 h incubation at 17 °C during the early stage of organogenesis. The motility, curvilinear velocity and straight-line velocity of fresh sperm was 93 ± 5%, 128 ± 13 μm/s and 89 ± 9 μm/s, respectively. There was a significant decrease of sperm motility rate between fresh sperm and cryopreserved sperm with/without addition of antifreeze proteins. The greatest motility among thawed samples was in the sperm cryopreserved with 10 μg/mL of AFPI (56 ± 20%), however, these data were not different compared to the sperm without antifreeze proteins (49 ± 14%). No statistical variations were detected in curvilinear velocity nor straight-line velocity. The fertilization rate with fresh sperm was 67 ± 7%. No significant differences were detected in fertilization rate between fresh and cryopreserved spermatozoa with/without addition of antifreeze proteins, except the sperm cryopreserved with 100 μg/mL of AFPIII (39 ± 14%). Thus, it is concluded that addition of antifreeze proteins to cryopreservation medium do not improve nor have toxicity effects on the quality and fertilization capacity of sterlet sperm after thawing.

Antifreeze Glycoproteins Bind Irreversibly to Ice.

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) inhibit ice growth via an adsorption-inhibition mechanism that assumes irreversible binding of AF(G)Ps to embryonic ice crystals and the inhibition of further growth. The irreversible binding of antifreeze glycoproteins (AFGPs) to ice has been questioned and remains poorly understood. Here, we used microfluidics and fluorescence microscopy to investigate the nature of the binding of small and large AFGP isoforms. We found that both AFGP isoforms bind irreversibly to ice, as evidenced by microfluidic solution exchange experiments. We measured the adsorption rate of the large AFGP isoform and found it to be 50% faster than that of AFP type III. We also found that the AFGP adsorption rate decreased by 65% in the presence of borate, a well-known inhibitor of AFGP activity. Our results demonstrate that the adsorption rate of AFGPs to ice is crucial for their ice growth inhibition capability.

PA-1 - Effects of supplementation of antifreeze proteins on follicular integrity of vitrified-warmed mouse ovary: comparison of two types of antifreeze proteins and their combination

PA-1 - Effects of supplementation of antifreeze proteins on follicular integrity of vitrified-warmed mouse ovary: comparison of two types of antifreeze proteins and their combination

III 型不凍タンパク質の低濃度水溶液中で成長する 氷結晶ベーサル面上のピット形成過程の観察

III 型不凍タンパク質の低濃度水溶液中で成長する 氷結晶ベーサル面上のピット形成過程の観察

Study of the plasma proteome of Atlantic cod (Gadus morhua): Effect of exposure to two PAHs and their corresponding diols

Occurrence of polycyclic aromatic hydrocarbon (PAH) contamination in the marine environment represents a risk to marine life and humans. In this study, plasma samples from Atlantic cod (Gadus morhua) were analysed by shotgun mass spectrometry to investigate the plasma proteome in response to exposure to single PAHs (naphthalene or chrysene) and their corresponding metabolites (dihydrodiols). In total, 369 proteins were identified and ranked according to their relative abundance. The levels of 12 proteins were found significantly altered in PAH exposed fish and are proposed as new biomarker candidates. Eleven proteins were upregulated, primarily immunoglobulin components, and one protein was downregulated (antifreeze protein type IV.) The uniformity of the upregulated proteins suggests a triggered immune response in the exposed fish.Overall, the results provide valuable knowledge for future studies of the Atlantic cod plasma proteome and generate grounds for establishing new plasma protein biomarkers for environmental monitoring of PAH related exposure.

Metabolic changes in droplet vitrified semen of wild endangered Persian sturgeon Acipenser persicus (Borodin, 1997)

Comparative quantitative metabolite profiling can be used for better understanding of cell functions and dysfunctions in particular circumstances such as sperm banking which is an important approach for cryopreservation of endangered species. Cryopreservation techniques have some deleterious effects on spermatozoa which put the obtained results in controversy. Therefore, in the present study, quantitative 1H NMR (Nuclear Magnetic Resonance) based metabolite profiling was conducted to evaluate metabolite changes related to energetics and some other detected metabolites in vitrified semen of critically endangered wild Acipenser persicus. The semen was diluted with extenders containing 0, 5, 10, and 15 μM of fish antifreeze protein (AFP) type III as a cryoprotectant. Semen-extenders were vitrified and stored for two days. Based on post-thaw motility duration and motility percentage assessments, two treatments with 10 μM and 0 μM of AFP had the highest and the lowest motility percentages respectively and they were objected to 1H NMR spectroscopy investigations in order to reveal the extremes of the metabolites dynamic range. Univariate (ANOVA) and multivariate (PCA) analysis of the resulting metabolic profiles indicated significant changes (P > 0.05) in metabolites. The level of some metabolites including acetate, adenine, creatine, creatine phosphate, lactate, betaine, sarcosine, β-alanine and trimethylamine N-oxide significantly decreased in vitrified semen while some others such as creatinine, guanidinoacetate, N, N-dimethylglycine, and glycine significantly increased. There were also significant differences between vitrified treatments in levels of creatine, creatine phosphate, creatinine, glucose, guanidinoacetate, lactate, N, N-dimethylglycine, and glycine, suggesting how fish AFP type III can be effective as a cryoprotectant.

Improvement in Ovarian Tissue Quality with Supplementation of Antifreeze Protein during Warming of Vitrified Mouse Ovarian Tissue.

Ice easily recrystallizes during warming after vitrification, and antifreeze protein (AFP) can inhibit the re-crystallization. However, no study has evaluated the effect of AFP treatment only thereon during warming. This study sought to compare AFP treatment protocols: a conventional protocol with AFP treatment during vitrification and first-step warming and a new protocol with AFP treatment during the first-step warming only. According to the protocols, 10 mg/mL of LeIBP (a type of AFP) was used. Five-week-old B6D2F1 mouse ovaries were randomly divided into a vitrified-warmed control and two experimental groups, one treated with the conventional AFP treatment protocol (LeIBP-all) and the other with the new AFP treatment protocol (LeIBP-w). For evaluation, ratios of ovarian follicle integrity, apoptosis, and DNA double-strand (DDS) damage/repairing were analyzed. The LeIBP-treated groups showed significantly higher intact follicle ratios than the control, and the results were similar between the LeIBP-treated groups. Apoptotic follicle ratios were significantly lower in both LeIBP-treated groups than the control, and the results were not significantly different between the LeIBP-treated groups. With regard to DDS damage/repairing follicle ratio, significantly lower ratios were recorded in both LeIBP-treated groups, compared to the control, and the results were similar between the LeIBP-treated groups. This study demonstrated that both protocols with LeIBP had a beneficial effect on maintaining follicle integrity and preventing follicle apoptosis and DDS damage. Moreover, the new protocol showed similar results to the conventional protocol. This new protocol could optimize the mouse ovary vitrification-warming procedure using AFP, while minimizing the treatment steps.

Solvation Shell Structure of Small Molecules and Proteins by IR-MCR Spectroscopy.

The flexibility of the hydrogen-bonded network of water is the basis for its excellent solvation properties. Accordingly, it is valuable to understand the properties of water in the solvation shell surrounding small molecules and biomolecules. Recent high-quality Raman spectra analyzed with Self-Modeling Curve Resolution (SMCR) have provided Raman spectra of small-molecule solvation shells. Here we apply SMCR to the complementary technique of Fourier transform infrared (FTIR) spectroscopy in the attenuated total reflection (ATR) configuration to extract the IR spectra of solvation shells. We first illustrate the method by obtaining the IR-MCR solvation shell spectra of tert-butanol (TBA), before applying it to antifreeze protein type III. Our results show that IR-SMCR spectroscopy is a powerful method for studying the solvation shell structure of small molecules and biomolecules. Given the wide availability of FTIR-ATR instruments, the method could prove to be an impactful tool for studying solvation and solvent-mediated interactions.

Effects of supplementation with antifreeze proteins on the follicular integrity of vitrified-warmed mouse ovaries: Comparison of two types of antifreeze proteins alone and in combination.

ObjectiveThe aim of this study was to analyze the effect of supplementing vitrification and warming solutions with two types of antifreeze proteins (AFPs) and the combination thereof on the follicular integrity of vitrified-warmed mouse ovaries.MethodsOvaries (n=154) were obtained from 5-week-old BDF1 female mice (n=77) and vitrified using ethylene glycol and dimethyl sulfoxide with the supplementation of 10 mg/mL of Flavobacterium frigoris ice-binding protein (FfIBP), 10 mg/mL of type III AFP, or the combination thereof. Ovarian sections were examined by light microscopy after hematoxylin and eosin staining, and follicular intactness was assessed as a whole and according to the type of follicle. Apoptosis within the follicles as a whole was detected by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay.ResultsThe proportion of overall intact follicles was significantly higher in the type III AFP-supplemented group (60.5%) and the combination group (62.9%) than in the non-supplemented controls (43.8%, p<0.05 for each). The proportion of intact primordial follicles was significantly higher in the FfIBP-supplemented (90.0%), type III AFP-supplemented (92.3%), and combination (89.7%) groups than in the non-supplemented control group (46.2%, p<0.05 for each). The proportions of non-apoptotic follicles were similar across the four groups.ConclusionSupplementation of the vitrification and warming solutions with FfIBP, type III AFP, or the combination thereof was equally beneficial for the preservation of primordial follicles in vitrified mouse ovaries.

The use of antifreeze protein type III for vitrification of in vitro matured bovine oocytes

The aim of this study was to evaluate the use of antifreeze protein type III (AFP III) into vitrification medium on meiotic spindle morphology of in vitro matured bovine oocytes as well as the fertilization and blastocyst rates. Mature cumulus-oocyte complexes (COC) were distributed in four groups: control (untreated), vitrified without supplementation (AFP0) or supplemented with 500 (AFP500) or 1000 ng/mL (AFP1000) into vitrification solutions. Samples from each group were used to analyze the organization of meiotic spindle by confocal microscopy and the remaining COC were submitted to in vitro fertilization and culture for eight days. Control group exhibited only 15% of abnormal spindle. However, the spindle morphology was affected in all vitrified groups regardless to AFP concentration: 75.8%, 76.1% and 69.2% (P > 0.05) for AFP0, AFP500 and AFP1000, respectively. Similar cleavage rate was obtained among the vitrified groups (AFP0 = 17.9%, AFP500 = 16.9% and AFP1000 = 17.8%), but lower (P < 0.05) compared with control group (68.7%). At Day 5 of culture, embryo production rate of AFP500 (30.8%) and AFP1000 (25.0%) were similar to control group (49.4%). However, at Day 8 of culture, AFP0, AFP500 and AFP1000 groups exhibited lower (P < 0.05) blastocyst rates (10.0%, 3.8% and 9.4%, respectively) when compared to control (41.1%). In conclusion, AFP III did not preserve meiotic spindle organization against the cryoinjuries. However, the use of AFP III improved embryo development at Day 5 of culture, although this effect was not maintained up to the blastocyst formation.

Pit Formation on the Basal Plane of Ice in Antifreeze Protein Type III Solution for Different Growth Mechanisms of Ice

When a single crystal of ice that has the basal plane several tenths of mm2 in area grows in a moderately active antifreeze protein (AFP) solution, numerous pits consisting of six pyramidal planes are formed on the basal plane. This pit formation suggests some interactions between the AFPs and the basal plane. In this study, we observed pit formation on the basal plane of ice growing in a 5 mg/mL fish AFP (type III) solution and examined three growth mechanisms (normal, spiral, and two-dimensional (2D) nucleation) of the basal plane by measuring the relationship between the growth rate and the degree of supercooling. We also measured the number density of pits in ice and found that the number density of pits for normal growth mode on molecularly rough surfaces was lower than that for spiral growth mode on relatively smooth surfaces, whereas pit formation was not observed during 2D nucleation growth mode. On the basis of these results, we proposed a model of pit formation during spiral growth mode. In this...

Influence of heating temperature, pressure and pH on recrystallization inhibition activity of antifreeze protein type III

The objective of this work was to investigate the influence of heating temperature, pressure and pH on ice recrystallization inhibition (IRI) activity and on the structure of fish antifreeze protein type III (AFP III). Results showed that temperature treatment up to 80 °C for one minute had no influence on IRI activity. 1H NMR spectroscopy demonstrated a fully reversible denaturation after temperature treatment at 80 °C up to 1 h. Adjusting pH with hydrochloric acid or sodium hydroxide increased the IRI activity independent of the pH value, whereas NMR experiments showed no significant structural change. Experiments with sodium chloride showed that the addition of ions is responsible for increased IRI activity and increasing ion concentration led to increased IRI activity up to a limiting value between 0.02 and 0.03 M. Pressure treatment up to 400 MPa for one minute had no influence on IRI activity.

Structure and collective dynamics of hydrated anti-freeze protein type III from 180 K to 298 K by X-ray diffraction and inelastic X-ray scattering.

We investigated hydrated antifreeze protein type III (AFP III) powder with a hydration level h (=mass of water/mass of protein) of 0.4 in the temperature range between 180 K and 298 K using X-ray diffraction and inelastic X-ray scattering (IXS). The X-ray diffraction data showed smooth, largely monotonic changes between 180 K and 298 K without freezing water. Meanwhile, the collective dynamics observed by IXS showed a strong change in the sound velocity at 180 K, after being largely temperature independent at higher temperatures (298-220 K). We interpret this change in terms of the dynamic transition previously discussed using other probes including THz IR absorption spectroscopy and incoherent elastic and quasi-elastic neutron scattering. This finding suggests that the dynamic transition of hydrated proteins is observable on the subpicosecond time scale as well as nano- and pico-second scales, both in collective dynamics from IXS and single particle dynamics from neutron scattering. Moreover, it is most likely that the dynamic transition of hydrated AFP III is not directly correlated with its hydration structure.

Protein–water dynamics in antifreeze protein III activity

We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.