Isolation and characterization of type I antifreeze proteins from cunner, Tautogolabrus adspersus, order Perciformes

Antifreeze proteins (AFPs) are produced by many species of teleost fish that inhabit potentially lethal ice-laden seawater and afford them protection from freezing. To date type I AFPs have been fully characterized in two teleost orders: Pleuronectiformes and Scorpaeniformes. In this study, we report the isolation and complete characterization of a type I AFP present in fish from a third order: cunner (Tautogolabrus adspersus), order Perciformes (family Labridae). This protein was purified from blood plasma and found to belong to what is now known as classical type I AFP with their small size (mass 4095.16 Da), alanine richness (> 57 mol%), high α-helicity (> 99%) with the ability to undergo reversible thermal denaturation, 11 amino acid (ThrX(10)) repeat regions within the primary structure, the capacity to impart a hexagonal bipyramidal shaping to ice crystals and the conservation of an ice-binding site found in many of the other type I AFPs. Partial de novo sequencing of the plasma AFP accounted for approximately half of the peptide mass. Sequencing of a combined liver and skin cDNA library indicated that the protein is produced without a signal sequence. In addition the translated product of the AFP cDNA suggests that it codes for the AFP isolated from plasma. These results further solidify the hypothesis that type I AFPs are multiphyletic in origin and suggest that they represent remarkable examples of convergent evolution within three orders of teleost fish.     

Isolation and purification of antifreeze proteins from skin tissues of snailfish, cunner and sea raven

Antifreeze proteins/polypeptides (AFPs), which are found in diverse species of marine fish, are grouped into four distinct classes (types I-IV). The discovery of skin-specific type I AFPs established that this class contains distinct isoforms, liver-type and skin-type, which are encoded by separate gene families. In this study, type I AFPs were isolated and partially characterized from skin tissues of Atlantic snailfish (Liparis atlanticus) and cunner (Tautogolabrus adspersus). Interestingly, evidence from this study indicates that snailfish type I AFPs synthesized in skin tissues are identical to those circulating in their blood plasma. Furthermore, type II AFPs that are identical to those expressed in liver for export into blood were purified from sea raven (Hemitripterus americanus) skin tissue extracts. It is clear that epithelial tissues are an important source for antifreeze expression to enhance the complement of AFPs that protect fish from freezing in extreme cold environments. In addition, the evidence generated in this study demonstrates that expression of AFPs in fish skin is a widespread phenomenon that is not limited to type I proteins.     

Intra- and interspecific allozymic variation in Liparis fabricii and Liparis gibbus (Teleostei,Liparididae) from Spitsbergen waters

Summary In the literature, arctic snailfish of the genus Liparis are often identified as L. liparis, primarily due to the wide morphological variation reported for this species. Recent morphological studies have, however, identified separate arctic Liparis species. Electrophoretic studies of the genetic relationship between two such species (L. fabricii and L. gibbus), from deepwater localities west of Spitsbergen revealed significant differences in allozyme mobility at a series of gene-loci and thus supported the evidence that they are valid species. Their extremely low intra-specific variability, with average heterozygosities well below one percent, is comparable to measures for other polar teleosts. No protein coding locus was found suitable for studies of possible population structuring of L. gibbus due to the very low frequencies of all but the commonest allele.     

Isolation and characterization of skin-type, type I antifreeze polypeptides from the longhorn sculpin, Myoxocephalus octodecemspinosus

The antifreeze polypeptides (AFPs) are found in several marine fish and have been grouped into four distinct biochemical classes (type I-IV). Recently, the new subclass of skin-type, type I AFPs that are produced intracellularly as mature polypeptides have been identified in the winter flounder (Pleuronectes americanus) and the shorthorn sculpin (Myoxocephalus scorpius). This study demonstrates the presence of skin-type AFPs in the longhorn sculpin (Myoxocephalus octodecemspinosus), which produces type IV serum AFPs. Using polymerase chain reaction-based methods, a clone that encoded for a type I AFP was identified. The clone lacked a signal sequence, indicating that the mature polypeptide is produced in the cytosol. A recombinant protein was produced in Escherichia coli and antifreeze activity was characterized. Four individual Ala-rich polypeptides with antifreeze activity were isolated from the skin tissue. One polypeptide was completely sequenced by tandem MS. This study provides the first evidence of a fish species that produces two different biochemical classes of antifreeze proteins (type I and type IV), and enforces the notion that skin-type AFPs are a widespread biological phenomenon in fish.     

Isolation and characterization of hemolymph antifreeze proteins from larvae of the longhorn beetle Rhagium inquisitor (L.)

We describe a simple and effective procedure to isolate antifreeze proteins (AFPs) from the hemolymph of larvae of the longhorn beetle Rhagium inquisitor, and present some characteristics of their structures. Several AFPs were isolated from the hemolymph of this species by heat and acid extraction followed by cation exchange. The hemolymph contains at least six AFPs ranging in size from 12.5 to 12.8 kDa. Of these, three were separated to purity by the ion exchange step, as indicated by mass spectrometry. The remaining three forms were further separated by size exclusion chromatography, but could not be isolated to purity. All AFPs in the hemolymph of this species appears to have isoelectric points above 8.00. The dominant form, RiAFP(H4), was purified by the ion exchange step. Its amino acid composition reveals a lower level of cysteine and a higher level of threonine, arginine, alanine and glycine than seen in other insect AFPs. Its trypsin fingerprint does not match that of any known protein. It interacts with ice both in the anionic and cationic state.     

Isolation of seven polymorphic microsatellites in Ophioblennius atlanticus atlanticus (Perciformes,Blenniidae)

We isolated and characterized seven polymorphic microsatellite loci of Ophioblennius atlanticus atlanticus (Valenciennes, 1836) using an optimized protocol to construct and screen a microsatellite‐enriched genomic library. The analysis of variability was performed in 16 specimens from Faial Island (Azores, Portugal). The mean number of alleles was 8.71 ± 2.43 and the level of expected heterozygosity ranged from 0.764 to 0.903. The total exclusionary probabilities using these loci for the first and the second parent were 0.985 and 0.998, respectively, suggesting that these microsatellites are a useful tool for large‐scale parentage analysis.     

Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

Heterologous expression,refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

Age and growth of early-life-stage Atlantic tarpon (Megalops atlanticus) from the northcentral Gulf of Mexico

Age and growth of early-life-stage Atlantic tarpon Megalops atlanticus collected from Mississippi coastal waters in the northcentral Gulf of Mexico (GOM) are described using otolith microstructure analysis. Tarpon leptocephali (n = 95, 16.0—27.8 mm standard length, L_S) collected from June throughOctober 2013—2018, ranged in age from 22 to 43 days (mean = 30.9 ± 0.5 days). Leptocephalus somatic growth rates ranged 0.46—1.24 mm day⁻¹ (mean = 0.76 ± 0.02 mm day⁻¹), and leptocephalus otolith growth rates ranged 1.78—3.97 μm day⁻¹ (mean = 2.58 ± 0.04 μm day⁻¹). Growth rates were inversely correlated to leptocephalus age, indicating the shrinkage phase associated with leptocephalus metamorphosis. Juvenile tarpon (n = 358, 50—359 mm fork length, L_F) were collected from August through December 2007—2018. Juveniles exhibited a positive allometric relationship (adjusted R² = 0.99, P < 0.001) between length and mass. The age of 100 juveniles (71—277 mm L_F) ranged from 76 to 174 days. Juvenile growth rate was estimated as 1.56 ± 0.11 mm day⁻¹. Significant (P < 0.001) linear relationships were found between juvenile age and otolith metrics, including otolith mass (R² = 0.81) and radius (R² = 0.68). Evaluation of the backcalculated hatch dates suggests that specimens in the collection hatched from late May through mid-September with slight peaks during July and August. A Rao's Spacing Test of Uniformity indicates the presence of significant lunar periodicity in leptocephalus hatch dates (n = 95, U = 250.1, P < 0.05), with 50% of the leptocephali hatched within 5 days (before or after) of the full moon. This study fills critical gaps in the scientific knowledge of tarpon and provides estimates of early-life-history metrics for an iconic game fish at the northernmost extent of its GOM range.     

Age/size relations and food of two snailfishes,Liparis gibbus and Careproctus reinhardii (Teleostei,Liparididae) from Spitsbergen coastal waters

Summary The snailfish family Liparididae was well represented in bottom trawl hauls (205–370 m) from the Spitsbergen area, with Liparis gibbus being the dominant species. Careproctus reinhardti was less numerous. The present paper is based on 539 specimens of C. gibbus (size range 6–25cm) and 120 specimens of L. reinhardti (size range 5–26 cm). Both species showed a rather similar size and age distribution. The stomach contents reveal that both snailfish species feed both benthically and pelagically. Crustaceans, especially amphipods and decapods, were the most common prey items. With increasing size of the fish, the decapod Pandalus borealis becomes more important, particularly for L. gibbus. Juvenile Liparis (1.3–4.3 cm in length) were found pelagically (35–200 m), and their diet mainly consisted of copepods and young hyperiid amphipods.     

Isolation and characterization of phytoferritin from pea (Pisum sativum) and Lentil (Lens esculenta).

Ferritin was isolated from the seeds of pea (Pisum sativum) and lentil (Lens esculenta). The homogeneity of the phytoferritins was established by polyacrylamide-gel electrophoresis. The subunit molecular weights were respectively 20 300 and 21 400 for hte pea and lentil proteins. A neutron low-angle scattering study established the molecular weight of the oligomer as 480 000 for pea apoferritin and 510 000 for lentil apoferritin. Although the quaternary structure of 24 polypeptide chains is preserved, the phytoferritins have a larger cavity in the interior than mammalian ferritins and can thus potentially store 1.2-1.4 times as much iron. The amino acid composition of the phytoferritins show some similarities to those of mammalian apoferritins; tryptic 'fingerprinting' reveals that there are many differences in the amino acid sequence of plant and mammalian apoferritins.     

Structural and functional characterization of a C-type lectin-like antifreeze protein from rainbow smelt (Osmerus mordax).

Antifreeze proteins (AFPs) are produced by several cold-water fish species. They depress physiological freezing temperatures by inhibiting growth of ice crystals and, in so doing, permit the survival of these fish in seawater cooler than their normal freezing temperatures. The type II AFP from rainbow smelt (Osmerus mordax), which is a member of the C-type lectin superfamily, was characterized in terms of its Ca2+-binding quaternary structure and the role of its single N-linked oligosaccharide. The protein core of the smelt AFP, shown through sequence homology to be a C-type lectin carbohydrate-recognition domain, was found to be protease resistant. Smelt AFP was also shown to bind Ca2+, as determined by ruthenium red staining and a conformational change on Ca2+ binding detected by intrinsic fluorescence. The N-linked oligosaccharide was found to have no effect on protease resistance, dimerization, or antifreeze activity. Thus its role, if any, in the antifreeze function of this protein remains unknown. Smelt AFP was also shown to be a true intermolecular dimer composed of two separate subunits. This dimerization did not require the presence of N-linked oligosaccharide or bound Ca2+. Smelt AFP dimerization has implications for the effective solution concentration and measurement of its activity. This finding may also lead to new interpretation of the mechanism of ice-growth inhibition by this AFP.     

Purification and characterization of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Four antifreeze proteins (AFPs) were purified from larvae of the beetle Dendroides canadensis. The AFPs are similar in amino acid compositions, having high contents of hydrophilic amino acids (45–55 mol%) and cysteine (16 mol% Cys). Approximately half of the Cys residues form disulfide bridges, and both the disulfide bridges and free sulfhydryls are essential for activity. The N-terminals of the AFPs are blocked. The pH optimum of the AFPs is 7.8, but major loss of activity occurred only at very high pH (12.0). The detergents SDS and Triton X-100 did not inactivate the AFPs. Circular dichroism spectra indicate the presence of both and secondary structures in the AFPs, in addition to a large random structure component.Abbreviations AFP antifreeze protein - CD circular dichroism - DTT dithiothreitol - HPLC high pressure liquid chromatography - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid     

The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures

To date, all attempts at fish embryo cryopreservation have failed. One of the main reasons for this to occur is the high chilling sensitivity reported in fish embryos thus emphasizing the need for further testing of different methods and alternative cryoprotective agents (CPAs) in order to improve our chances to succeed in this purpose. In this work we have used the antifreeze protein type I (AFP I) as a natural CPA. This protein is naturally expressed in sub-arctic fish species, and inhibits the growth of ice crystals as well as recrystallization during thawing. Embryos from Sparus aurata were microinjected with AFP I at different developmental stages, 2 cells and blastula, into the blastomere-yolk interface and into the yolk sac, respectively. Control, punctured and microinjected embryos were subjected to chilling at two different temperatures, 0 degrees C (1h) and -10 degrees C (15min) when embryos reached 5-somite stage. Embryos were subjected to -10 degrees C chilling in a 3M DMSO extender to avoid ice crystal formation in the external solution. Survival after chilling was established as the percentage of embryos that hatch. To study the AFP I distribution in the microinjected embryos, a confocal microscopy study was done. Results demonstrate that AFP I can significantly improve chilling resistance at 0 degrees C, particularly in 2-cell microinjected embryos, displaying nearly 100% hatching rates. This fact is in agreement with the confocal microscopy observations which confirmed the presence of the AFP protein in embryonic cells. These results support the hypothesis that AFP protect cellular structures by stabilizing cellular membranes.