Molecular structure of a hyperactive antifreeze protein adsorbed to ice.

Antifreeze proteins (AFPs) are a unique class of proteins that bind to ice crystal surfaces and arrest their growth. The working mechanism of AFPs is not well understood because, as of yet, it was not possible to perform molecular-scale studies of AFPs adsorbed to the surface of ice. Here, we study the structural properties of an AFP from the insect Rhagium mordax (RmAFP) adsorbed to ice with surface specific heterodyne-detected vibrational sum-frequency generation spectroscopy and molecular dynamic simulations. We find that RmAFP, unlike other proteins, retains its hydrating water molecules upon adsorption to the ice surface. This hydration water has an orientation and hydrogen-bond structure different from the ice surface, thereby inhibiting the insertion of water layers in between the protein and the ice surface.

Detecting seasonal variation of antifreeze protein distribution in Rhagium mordax using immunofluorescence and high resolution microscopy.

Larvae of the blackspotted pliers support beetle, Rhagium mordax, were collected monthly, for the duration of 2012 and fixed. The larvae were embedded in paraffin wax and sectioned. Using fluorophore-coupled antibodies specific to the R. mordax antifreeze protein, RmAFP1, sections were visualised with UV reflected light microscopy. An automated software analysis method was developed in order to discard autofluorescence, and quantify fluorescence from bound antibodies. The results show that R. mordax cuticle and gut exhibit a higher degree of fluorophore-bound fluorescence during summer, than in the cold months. It is hypothesised that R. mordax stores RmAFP1 in, or near, the fat body during times when freeze avoidance is not needed.

An open source cryostage and software analysis method for detection of antifreeze activity.

The aim of this study is to provide the reader with a simple setup that can detect antifreeze proteins (AFP) by inhibition of ice recrystallisation in very small sample sizes. This includes an open source cryostage, a method for preparing and loading samples as well as a software analysis method. The entire setup was tested using hyperactive AFP from the cerambycid beetle, Rhagium mordax. Samples containing AFP were compared to buffer samples, and the results are visualised as crystal radius evolution over time and in absolute change over 30 min. Statistical analysis showed that samples containing AFP could reliably be told apart from controls after only two minutes of recrystallisation. The goal of providing a fast, cheap and easy method for detecting antifreeze proteins in solution was met, and further development of the system can be followed at https://github.com/pechano/cryostage.

Excess post-hypoxic oxygen consumption is independent from lactate accumulation in two cyprinid fishes.

Carassius carassius responds to hypoxic conditions by conversion of lactate into ethanol, which is excreted over the gills. However, a closely related species, Cyprinus carpio, does not possess the ability to produce ethanol and would be expected to accumulate lactate during hypoxic exposure. While the increase in oxygen consumption in fish required following strenuous exercise or low environmental oxygen availability has been frequently considered, the primary contributing mechanism remains unknown. This study utilized the close relationship but strongly divergent physiology between C. carpio and C. carassius to examine the possible correlation between excess post-hypoxic oxygen consumption (EPHOC) and lactate accumulation. No difference in the EPHOC:O2 deficit ratio was observed between the two species after 2.5h anoxia, with ratios of 2.0±0.6 (C. carpio) and 1.3±0.3 (C. carassius). As predicted, lactate accumulation dynamics did significantly differ between the species in both plasma and white muscle following anoxic exposure. Significant lactate accumulation was seen in both plasma and muscle in C. carpio, but there was no accumulation of lactate in white muscle tissue of C. carassius. These findings indicate that lactate accumulated as a consequence of 2.5h anoxic exposure is not a major determinant of the resulting EPHOC.

Survival in extreme environments - on the current knowledge of adaptations in tardigrades.

Tardigrades are microscopic animals found worldwide in aquatic as well as terrestrial ecosystems. They belong to the invertebrate superclade Ecdysozoa, as do the two major invertebrate model organisms: Caenorhabditis elegans and Drosophila melanogaster. We present a brief description of the tardigrades and highlight species that are currently used as models for physiological and molecular investigations. Tardigrades are uniquely adapted to a range of environmental extremes. Cryptobiosis, currently referred to as a reversible ametabolic state induced by e.g. desiccation, is common especially among limno-terrestrial species. It has been shown that the entry and exit of cryptobiosis may involve synthesis of bioprotectants in the form of selective carbohydrates and proteins as well as high levels of antioxidant enzymes and other free radical scavengers. However, at present a general scheme of mechanisms explaining this phenomenon is lacking. Importantly, recent research has shown that tardigrades even in their active states may be extremely tolerant to environmental stress, handling extreme levels of ionizing radiation, large fluctuation in external salinity and avoiding freezing by supercooling to below -20 °C, presumably relying on efficient DNA repair mechanisms and osmoregulation. This review summarizes the current knowledge on adaptations found among tardigrades, and presents new data on tardigrade cell numbers and osmoregulation.

Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor.

Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs and it is structurally unique in several aspects. In contrast to the high content of disulfide bond-formation observed in other coleopteran AFPs, RiAFP contains only a single such bond. Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part of these repeat segments is preserved as TxTxTxT, which is effectively an expansion of the TxT ice-binding motif found in the AFPs of several known insect AFPs.

Ice-active proteins and cryoprotectants from the New Zealand alpine cockroach, Celatoblatta quinquemaculata.

Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (approximately 2 degrees C) and the gut contents somewhat lower levels (approximately 0.6 degrees C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization.

Seasonal variations in antifreeze protein activity and haemolymph osmolality in larvae of the beetle Ragium mordax (Coleoptera: Cerambycidae).

Larvae of Rhagium mordax empty their guts in preparation for the winter, which alone may enable the larvae to supercool down to -20 degree C or below. This should be sufficient for the larvae to over winter in Denmark if they can prevent inoculation. Antifreeze proteins (AFP) prevent inoculation in adult Rhagium inquisitor and this is also likely in the larvae of R. mordax, as they are in contact with ice in their hibernacula during the winter. arvae of R. mordax probably produce AFPs in the early autumn, however, in some individuals thermal hysteresis (TH) as high as 5.01 degree C was observed in June. Whether or not these individuals have a constant level of TH in their haemolymph all year or if they produce further antifreeze proteins during the autumn is unknown. The lowest measured in January was 7.49 degree C (the highest during this month was 9.08 degree C) so it is likely that the individuals with the highest TH in June also produce AFPs. Haemolymph osmolality in R. mordax is relatively low compared to other freeze avoiding insects, samples taken in January peak at 741 mOsm (+/-127 mOsm). The results of this study are compared with similar data for the closely related R. inquisitor.

Effects of osmolytes on hexokinase kinetics combined with macromolecular crowding: test of the osmolyte compatibility hypothesis towards crowded systems.

We investigated the effect of compatible and non-compatible osmolytes in combination with macromolecular crowding on the kinetics of yeast hexokinase. This was motivated by the fact that almost all studies concerning the osmolyte effects on enzyme activity have been performed in diluted buffer systems, which are far from the physiological conditions within cells, where the cytosol contains several hundred mg protein ml(-1). Four organic (glycerol, betaine, TMAO and urea) and one inorganic (NaCl) osmolyte were tested. It was concluded that the effect of compatible osmolytes (glycerol, betaine and TMAO) on V(max) and K(M) was practically equivalent in pure buffer and in 200-250 mg BSA ml(-1) supporting the view that these small organic osmolytes do minimal perturbance on enzyme function in physiological solutions. The effect of urea on enzyme kinetics was not independent of protein concentration, since the presence of 250 mg BSA ml(-1) partly compensated the perturbing effect of urea. Even though the organic osmolytes glycerol, betaine and TMAO are generally considered compatible with enzyme function, especially glycerol did have a significant effect on hexokinase kinetics, decreasing both k(cat), K(M) and k(cat)/K(M). The osmolytes decreased k(cat)/K(M) in the order: NaCl>Urea>TMAO/glycerol>betaine. For the organic osmolytes this order correlates with the degree of exclusion from protein-water interfaces. Thus, the stronger the exclusion the weaker the perturbing effects on k(cat)/K(M).

Purification and structural analysis of a type III antifreeze protein from the european eelpout Zoarces viviparus.

It has recently been reported that the eelpout Zoarces viviparus synthesizes a family of antifreeze proteins (AFP) similar in sequence to type III AFPs. A method has been set up to separate these antifreeze proteins from blood serum of this teleost species. A total of nine proteins with antifreeze activity have been isolated, several to a purity suited for NMR experiments. One of the proteins, Zvafp13, has been subject to partial structure determination by NMR. 1D- and 2D-H NMR analyses were carried out. In the 1D-experiments it was observed that the protein contained 28 slow-exchanging amides, suggesting a compact structure. The 2D-experiments were utilized to assign observed signals to specific amino acids. From TOCSY- and NOESY-experiments 35 out of a total of 66 amino acids were assigned. The amide exchange pattern, protein primary sequence, chemical shifts and NOE-cross-peaks between amides and -protons in the -sheets suggest that Zvafp13 structurally resembles the recombinant type III AFP rQAE m1.1.

Isolation and some characterisation of antifreeze protein from the European eelpout Zoarces viviparus.

The European eelpout Zoarces viviparus is a common inhabitant in the coastal areas of the eastern Atlantic Ocean and the Baltic region. At least 3 different antifreeze proteins were purified from Z. viviparus serum but more isoforms are most likely present. Two antifreeze proteins with molecular weights of approx. 6.5-7 kDa were characterised and found to share high similarity to the type III antifreeze proteins found in other members of the family Zoarcidae. The antifreeze activity of Z. viviparus antifreeze proteins is concentration dependent and showed a saturation effect when the protein concentration reached 30 mg.ml-1 (crude serum) and 8 mg.ml-1 (partly purified serum) respectively. Further the antifreeze activity was found to be dependent of the buffer osmolality resulting in increasing thermal hysteresis when buffer osmolality was raised from 0 to 1M.

Ice-active proteins from the Antarctic nematode Panagrolaimus davidi.

The Antarctic nematode Panagrolaimus davidi has an ice-active protein that shows recrystallization inhibition but no thermal hysteresis. It belongs to a class of ice-active proteins found in a variety of freezing-tolerant organisms that display insignificant levels of thermal hysteresis in the context of the environmental temperatures to which they are exposed. The recrystallization inhibition activity of the P. davidi ice-active protein is present at low concentrations, is relatively heat stable, is affected more by acid than by alkaline pH, is not calcium dependant and is not affected by reagents that target carbohydrate residues or sulphydryl linkages. A hexagonal ice crystal growth form also indicates the presence of an ice-active protein. This protein could have important functions in the survival of intracellular freezing by this organism by controlling the stability of ice after its formation.

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.

Variations in antifreeze activity and serum inorganic ions in the eelpout Zoarces viviparus: antifreeze activity in the embryonic state.

The eelpout Zoarces viviparus is a common inhabitant in the shallow waters along the Danish coastline. Specimens were caught in the brackish (12-16 per thousand) Roskilde fjord where water temperatures range from >20 degrees C during summer to subzero in winter. The serum melting points found in Z. viviparus varied between -0.76 (September) to -0.94 degrees C (January). Eighty to 97% of the serum melting points could be attributed to sodium, chloride and potassium. Hysteresis freezing points showed seasonal variation varying from -0.83 (September) to -2.08 degrees C (February). Serum antifreeze activity showed a seasonal variation with high levels (>1.2 degrees C) in winter and low levels (<0.1 degrees C) during summer and autumn. Antifreeze proteins are responsible for this antifreeze activity. Antifreeze activity was also found in Z. viviparus during their embryological development in the female ovary. Embryo thermal hysteresis reached the maximum level (approx. 0.6 degrees C) during December and maintained this level until parturition in January. Antifreeze activity seems unaffected by diminishing ice crystal fractions at ice fractions below 0.1 whereas ice fractions above 0.1 caused a decline in antifreeze activity.

Extreme resistance to desiccation in overwintering larvae of the gall fly Eurosta solidaginis (Diptera, tephritidae).

During winter, larvae of the goldenrod gall fly Eurosta solidaginis are exposed for extended periods to severe low ambient temperatures and low humidities within plant galls. The resistance of these larvae to desiccation at various temperatures and humidities, the transition (critical) temperature, and the effects of treatment with organic solvents on the larval rates of water loss and on changes in osmolality during desiccation were examined. The water loss rates of the flesh fly Sarcophaga crassipalpis under desiccating conditions were also measured. The water permeability of the cuticle of E. solidaginis larvae was very low (0.038 microgram h(-1 )cm(-2 )Pa(-1) at 20 C and 4% relative humidity) compared with that of larvae of other species. The value for E. solidaginis is equivalent to that of the very drought-resistant larvae of the tenebrionid beetle Tenebrio molitor (0.038 microgram h(-1)cm(-2)Pa(-1) at 30 C). In contrast, the permeability of larvae of the flesh fly Sarcophaga crassipalpis at 20 C and 4 % relative humidity was 0.331 microgram h(-1) cm(-2)Pa(-1). The thermal dependence of the cuticular permeability increased with temperature by approximately 0.0010 +microgram h(-1) cm(-2)Pa(-1) C(-1) in the interval between 4 and 40 C. At the transition temperature of 40 C, the thermal dependence of the permeability increased abruptly to 0.0400 microgram h(-1)cm(-2)Pa(-1) C(-1). Larvae treated with hexane and acetone remained remarkably resistant to water loss. However, treatment with chloroform:methanol increased the water loss rate approximately 25-fold. During desiccation at 4 C and 4% relative humidity for 21 days, E. solidaginis larvae showed a mass loss of 18.5+/-4.4 % (mean +/- s.e.m., N=6). Animals dried under the same conditions over the same period showed a haemolymph osmolality of 851+/-75 mosmol kg(-1) (N=4). Larvae freshly removed from the galls showed a haemolymph osmolality of 918+/-67 mosmol kg(-1)(N=3). A higher osmolality in the dried compared with the fresh larvae would have been expected. The present observation suggests that important ions in the haemolymph may have been excreted or rendered osmotically inactive during desiccation.

Aspects of natural cold tolerance in ectothermic animals.

Polar, alpine and temperate ectothermic (cold-blooded) animals encounter temperatures below the melting point of their body fluids either diurnally or seasonally. These animals have developed a number of biochemical and physiological adaptations to survive the low temperatures. The problems posed to the animals during cold periods include changes in membrane and protein structure due to phase changes in these molecules, changes in electrolyte concentrations and other solutes in the body fluids as well as changes in metabolism. Cold-tolerant ectothermic animals can be divided into two groups depending which of two 'strategies' they employ to survive the low temperatures: freeze-tolerant animals which survive ice formation in the tissues and freeze-avoiding animals which tolerate the low temperatures but not crystallization of the body fluids. The adaptations are mainly directed towards the control or avoidance of ice formation and include the synthesis of low mol. wt cryoprotectants, ice-nucleating agents and antifreeze proteins. However, some of the adaptations such as the synthesis of low mol. wt cryoprotectants are also more specific in their mechanism, e.g. direct stabilizing interaction with membranes and proteins. The mechanisms employed by such animals may offer ideas and information on alternative approaches which might be usefully employed in the cryopreservation of cells and tissues frequently required in assisted reproductive technology.

Recrystallization in a freezing tolerant Antarctic nematode, Panagrolaimus davidi, and an alpine weta, Hemideina maori (Orthoptera; Stenopelmatidae).

The ability of haemolymph from the freezing tolerant weta, Hemideina maori, and supernatant from homogenates of the freezing tolerant nematode Panagrolaimus davidi to inhibit the recrystallization of ice was examined using the "splat freezing" technique and annealing on a cryomicroscope stage. There was no recrystallization inhibition in weta haemolymph or in insect ringer controls. Recrystallization inhibition was present in the nematode supernatant but this was destroyed by heating and was absent in controls. P. davidi survives intracellular freezing and recrystallization inhibition may be important for the survival of this stress.