Subzero nonfreezing cryopresevation of rat hearts using antifreeze protein I and antifreeze protein III

The purpose of the present study was to evaluate whether AFPs protect the heart from freezing and improve survival and viability in subzero cryopreservation. Hearts were subject to 5 preservation protocols; University of Wisconsin solution (UW) at 4 degrees C, UW at -1.3 degrees C without nucleation, UW at -1.3 degrees C with nucleation, UW AFP I (15 mg/cm(3)) at -1.3 degrees C with nucleation, and in UW AFP III (15 mg/cm(3)) at -1.3 degrees C with nucleation. Hearts were preserved for 24, 28, and 32 h, rewarmed and connected to the working isolated perfusion system. Data [heart rate (HR), coronary flow (CF), and developed pressure (dP)] was collected 30 and 60 min after reperfusion. Hearts preserved at -1.3 degrees C without AFPs froze, while hearts preserved with AFP did not freeze when nucleation was initiated and survived. Survival and dP of hearts preserved for 24h at -1.3 degrees C using AFP III was better than those preserved at 4 degrees C, (dP; 1.4 vs. 0.8, p<0.05). Four of six hearts and six of six hearts died when preserved at 4 degrees C for 28 and 32 h, respectively, all of the hearts that were preserved at -1.3 degrees C with or without AFPs survived after 28 h (n=18) and 32 h (n=18). CF was higher in UW -1.3 degrees C group without attempted nucleation than in AFP I and AFP III groups after 28 and 32 h (3.4 vs. 1.7, p<0.05, and 3.4 vs. 1.7, p<0.05, respectively). In conclusion, AFPs were found to protect the heart from freezing and improve survival and dP (AFP III) in prolonged subzero preservation.     

Cooperative function of ammonium polyacrylate with antifreeze protein type I

Activity of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) is often determined by thermal hysteresis, which is the difference between the melting temperature and the nonequilibrium freezing temperature of ice in AF(G)P solutions. In this study, we confirmed that thermal hysteresis of AFP type I is significantly enhanced by a cooperative function of ammonium polyacrylate (NH4PA). Thermal hysteresis of mixtures of AFP type I and NH4PA was much larger than the sum of each thermal hysteresis of AFP type I and NH4PA alone. In mixed solutions of AFP type I and NH4PA in the thermal hysteresis region, hexagonal pyramidal-shaped pits densely formed on ice surfaces close to the basal planes. The experimental results suggest that the cooperative function of NH4PA with AFP type I was caused either by the increase in adsorption sites of AFP type I on ice or by the adsorption of AFP type I aggregates on ice.     

Solution structure of a recombinant type I sculpin antifreeze protein

We have determined the solution structure of rSS3, a recombinant form of the type I shorthorn sculpin antifreeze protein (AFP), at 278 and 268 K. This AFP contains an unusual sequence of N-terminal residues, together with two of the 11-residue repeats that are characteristic of the type I winter flounder AFP. The solution conformation of the N-terminal region of the sculpin AFP has been assumed to be the critical factor that results in recognition of different ice planes by the sculpin and flounder AFPs. At 278 K, the two repeats units (residues 11-20 and 21-32) in rSS3 form a continuous alpha-helix, with the residues 30-33 in the second repeat somewhat less well defined. Within the N-terminal region, residues 2-6 are well defined and helical and linked to the main helix by a more flexible region comprising residues A7-T11. At 268 K the AFP is overall more helical but retains the apparent hinge region. The helical conformation of the two repeats units is almost identical to the corresponding repeats in the type I winter flounder AFP. We also show that while tetracetylated rSS3 has antifreeze activity comparable to the natural AFP, its overall structure is the same as that of the unacetylated peptide. These data provide some insight into the structural determinants of antifreeze activity and should assist in the development of models that explain the recognition of different ice interfaces by the sculpin and flounder type I AFPs.     

Activity of a two-domain antifreeze protein is not dependent on linker sequence

The reported NMR structure of RD3, a naturally occurring two-domain antifreeze protein, suggests that the two nearly identical domains are oriented to allow simultaneous binding of their active regions to the ice surface. It is implied that the nine residues linking the two domains play a role in this alignment, but this has not been established. We have designed and expressed a modified form of RD3 that replaces the nine-residue linker with a generic sequence of one serine and eight glycine residues to test the importance of the linker amino acid sequence. The modified linker is shown to have significantly different characteristics compared to the original linker. Heteronuclear nuclear Overhauser effect experiments show that the new linker residues have more mobility than the linker residues in the native protein. Further, NMR data show that the folding of the C-terminal domain is somewhat perturbed by the altered linker. Finally, distributions of residual dipolar couplings indicate that the two domains tumble and move independently of each other. Nevertheless, the thermal hysteresis activity of the modified protein is indistinguishable from that of native RD3, proving that increased activity of the two-domain antifreeze protein is not dependent on structure of the linker.     

Structure of type I antifreeze protein and mutants in supercooled water

Many organisms are able to survive subzero temperatures at which bodily fluids would normally be expected to freeze. These organisms have adapted to these lower temperatures by synthesizing antifreeze proteins (AFPs), capable of binding to ice, which make further growth of ice energetically unfavorable. To date, the structures of five AFPs have been determined, and they show considerable sequence and structural diversity. The type I AFP reveals a single 37-residue alpha-helical structure. We have studied the behavior of wild-type type I AFP and two "inactive" mutants (Ala17Leu and Thr13Ser/Thr24Ser) in normal and supercooled solutions of H(2)O and deuterium oxide (D(2)O) to see if the structure at temperatures below the equilibrium freezing point is different from the structure observed at above freezing temperatures. Analysis of 1D (1)H- and (13)C-NMR spectra illustrate that all three proteins remain folded as the temperature is lowered and even seem to become more alpha-helical as evidenced by (13)C(alpha)-NMR chemical shift changes. Furthermore, (13)C-T(2) NMR relaxation measurements demonstrate that the rotational correlation times of all three proteins behave in a predictable manner under all temperatures and conditions studied. These data have important implications for the structure of the AFP bound to ice as well as the mechanisms for ice-binding and protein oligomerization.     

Type I shorthorn sculpin antifreeze protein: recombinant synthesis, solution conformation, and ice growth inhibition studies

A number of structurally diverse classes of "antifreeze" proteins that allow fish to survive in sub-zero ice-laden waters have been isolated from the blood plasma of cold water teleosts. However, despite receiving a great deal of attention, the one or more mechanisms through which these proteins act are not fully understood. In this report we have synthesized a type I antifreeze polypeptide (AFP) from the shorthorn sculpin Myoxocephalus scorpius using recombinant methods. Construction of a synthetic gene with optimized codon usage and expression as a glutathione S-transferase fusion protein followed by purification yielded milligram amounts of polypeptide with two extra residues appended to the N terminus. Circular dichroism and NMR experiments, including residual dipolar coupling measurements on a 15N-labeled recombinant polypeptide, show that the polypeptides are alpha-helical with the first four residues being more flexible than the remainder of the sequence. Both the recombinant and synthetic polypeptides modify ice growth, forming facetted crystals just below the freezing point, but display negligible thermal hysteresis. Acetylation of Lys-10, Lys-20, and Lys-21 as well as the N terminus of the recombinant polypeptide gave a derivative that displays both thermal hysteresis (0.4 degrees C at 15 mg/ml) and ice crystal faceting. These results confirm that the N terminus of wild-type polypeptide is functionally important and support our previously proposed mechanism for all type I proteins, in which the hydrophobic face is oriented toward the ice at the ice/water interface.     

Ice nucleation inhibition: mechanism of antifreeze by antifreeze protein

The effect of antifreeze protein type III (one type of fish antifreeze protein) on ice crystallization was examined quantitatively based on a "micro-sized ice nucleation" technique. It was found for the first time that antifreeze proteins can inhibit the ice nucleation process by adsorbing onto both the surfaces of ice nuclei and dust particles. This leads to an increase of the ice nucleation barrier and the desolvation kink kinetics barrier, respectively. Based on the latest nucleation model, the increases in the ice nucleation barrier and the kink kinetics barrier were measured. This enables us to quantitatively examine the antifreeze mechanism of antifreeze proteins for the first time.     

Conjugation of type I antifreeze protein to polyallylamine increases thermal hysteresis activity

Antifreeze proteins (AFPs) are ice binding proteins found in some plants, insects, and Antarctic fish allowing them to survive at subzero temperatures by inhibiting ice crystal growth. The interaction of AFPs with ice crystals results in a difference between the freezing and melting temperatures, termed thermal hysteresis, which is the most common measure of AFP activity. Creating antifreeze protein constructs that reduce the concentration of protein needed to observe thermal hysteresis activities would be beneficial for diverse applications including cold storage of cells or tissues, ice slurries used in refrigeration systems, and food storage. We demonstrate that conjugating multiple type I AFPs to a polyallylamine chain increases thermal hysteresis activity compared to the original protein. The reaction product is approximately twice as active when compared to the same concentration of free proteins, yielding 0.5 °C thermal hysteresis activity at 0.3 mM protein concentration. More impressively, the amount of protein required to achieve a thermal hysteresis of 0.3 °C is about 100 times lower when conjugated to the polymer (3 μM) compared to free protein (300 μM). Ice crystal morphologies observed in the presence of the reaction product are comparable to those of the protein used in the conjugation reaction.     

Accumulation of type I fish antifreeze protein in transgenic tobacco is cold-specific

Expression of fish antifreeze protein (AFP) genes in plants is a possible means of increasing their frost resistance and freeze tolerance. Initial work involved transfer into tobacco of an AFP gene from winter flounder which codes for the alanine-rich, -helical Type I AFP. Plants were transformed with a gene construct in which the preproAFP cDNA was inserted between the cauliflower mosaic virus 19S RNA promoter and the nopaline synthetase polyadenylation site. Although transgenic plants produced AFP mRNA, no AFP was detected on western blots. Re-evaluation of AFP expression in these transgenic plants showed that AFP accumulated to detectable levels only after exposure of the plant to cold. Extracts of plants incubated at 4°C for 24 h contained a protein which co-migrated with winter flounder proAFP and was cross-reactive to Type I AFP antisera. Two other minor protein bands of slightly higher apparent M _r also cross-reacted with the antisera and are thought to represent processing intermediates. The proAFP was unique to the transgenic plants and was absent in extracts taken prior to cold exposure. AFP levels increased over the first 48 h of cold incubation then remained stable. Since the -helix content of Type I AFP has been shown to decrease markedly at warmer temperatures, we postulate that Type I AFP stability in transgenic plants is dependent on its secondary structure.     

Type I antifreeze proteins expressed in snailfish skin are identical to their plasma counterparts

Type I antifreeze proteins (AFPs) are usually small, Ala-rich alpha-helical polypeptides found in right-eyed flounders and certain species of sculpin. These proteins are divided into two distinct subclasses, liver type and skin type, which are encoded by separate gene families. Blood plasma from Atlantic (Liparis atlanticus) and dusky (Liparis gibbus) snailfish contain type I AFPs that are significantly larger than all previously described type I AFPs. In this study, full-length cDNA clones that encode snailfish type I AFPs expressed in skin tissues were generated using a combination of library screening and PCR-based methods. The skin clones, which lack both signal and pro-sequences, produce proteins that are identical to circulating plasma AFPs. Although all fish examined consistently express antifreeze mRNA in skin tissue, there is extreme individual variation in liver expression - an unusual phenomenon that has never been reported previously. Furthermore, genomic Southern blot analysis revealed that snailfish AFPs are products of multigene families that consist of up to 10 gene copies per genome. The 113-residue snailfish AFPs do not contain any obvious amino acid repeats or continuous hydrophobic face which typify the structure of most other type I AFPs. These structural differences might have implications for their ice-crystal binding properties. These results are the first to demonstrate a dual liver/skin role of identical type I AFP expression which may represent an evolutionary intermediate prior to divergence into distinct gene families.     

Polycarboxylates enhance beetle antifreeze protein activity

Antifreeze proteins (AFPs) lower the noncolligative freezing point of water in the presence of ice below the ice melting point. The temperature difference between the melting point and the noncolligative freezing point is termed thermal hysteresis (TH). The magnitude of the TH depends on the specific activity and the concentration of AFP, and the concentration of enhancers in the solution. Known enhancers are certain low molecular mass molecules and proteins. Here, we investigated a series of polycarboxylates that enhance the TH activity of an AFP from the beetle Dendroides canadensis (DAFP) using differential scanning calorimetry (DSC). Triethylenetetramine-N,N,N',N',N',N'-hexaacetate, the most efficient enhancer identified in this work, can increase the TH of DAFP by nearly 1.5 fold over than that of the published best enhancer, citrate. The Zn(2+) coordinated carboxylate results in loss of the enhancement ability of the carboxylate on antifreeze activity. There is not an additional increase in TH when a weaker enhancer is added to a stronger enhancer solution. These observations suggest that the more carboxylate groups per enhancer molecule the better the efficiency of the enhancer and that the freedom of motion of these molecules is necessary for them to serve as enhancers for AFP. The hydroxyl groups in the enhancer molecules can also positively affect their TH enhancement efficiency, though not as strongly as carboxylate groups. Mechanisms are discussed.     

Type II fish antifreeze protein accumulation in transgenic tobacco does not confer frost resistance

Type II fish antifreeze protein (AFP) is active in both freezing point depression and the inhibition of ice recrystallization. This extensively disulfidebonded 14 kDa protein was targeted for accumulation in its pro and mature forms in the cytosol and apoplast of transgenic tobacco plants. Type II AFP gene constructs under control of a duplicate cauliflower mosaic virus 35S promoter, both with and without a native plant transit peptide sequence, were introduced into tobacco by Agrobacterium tumefaciensmediated transformation. AFP did not accumulate in the cytosol of transgenic plants, but active AFP was present as 2% of the total protein present in the apoplast. Plantproduced AFP was the same size as mature Type II AFP isolated from fish, and was comparable to wildtype AFP in thermal hysteresis activity and its effect on ice crystal morphology. Field trials conducted in late summer on R1 generation transgenic plants showed similar AFP accumulation in plants under field conditions at levels suitable for largescale production: but no difference in frost resistance was observed between transgenic and wildtype plants during the onset of early fall frosts.     

Structural modeling of snow flea antifreeze protein

The glycine-rich antifreeze protein recently discovered in snow fleas exhibits strong freezing point depression activity without significantly changing the melting point of its solution (thermal hysteresis). BLAST searches did not detect any protein with significant similarity in current databases. Based on its circular dichroism spectrum, discontinuities in its tripeptide repeat pattern, and intramolecular disulfide bonding, a detailed theoretical model is proposed for the 6.5-kDa isoform. In the model, the 81-residue protein is organized into a bundle of six short polyproline type II helices connected (with one exception) by proline-containing turns. This structure forms two sheets of three parallel helices, oriented antiparallel to each other. The central helices are particularly rich in glycines that facilitate backbone carbonyl-amide hydrogen bonding to four neighboring helices. The modeled structure has similarities to polyglycine II proposed by Crick and Rich in 1955 and is a close match to the polyproline type II antiparallel sheet structure determined by Traub in 1969 for (Pro-Gly-Gly)_n. Whereas the latter two structures are formed by intermolecular interactions, the snow flea antifreeze is stabilized by intramolecular interactions between the helices facilitated by the regularly spaced turns and disulfide bonds. Like several other antifreeze proteins, this modeled protein is amphipathic with a putative hydrophobic ice-binding face.     

抗冻蛋白的特性和作用机制

抗冻蛋白是非常热门的研究领域,具有广阔的应用前景。本文结合最新的报道,从抗冻蛋白的多样性、热带活性和改变冰的生长,综述了抗冻蛋白的特性,阐释了抗冻蛋白的作用机制的三种假说：“偶极子—偶极子”假说、氢原子结合模型假说和刚体能量学说。     

抗冻蛋白的研究进展

本文概述了抗冻蛋白的种类、生成和性质,总结了抗冻蛋白基因及基因转化方面的研究情况。     

Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants

Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly expressed in Escherichia coli strain BL21 (DE3). The activity of the purified and refolded recombinant proteins was analyzed by measurement of thermal hysteresis (TH) activity and detection of in vitro antifreeze activity by measuring enhanced cold resistance of bacteria. Two carrot AFP mutants generated by site-directed mutagenesis were also expressed and purified under these conditions for use in parallel experiments. Recombinant DcAFP displayed a TH activity equivalent to that of native DcAFP, while mutants DcAFP-N130Q and rDcAFP-N130V showed 32 and 43% decreases in TH activity, respectively. Both the recombinant DcAFP and its mutants were able to enhance the cold resistance of bacteria, to degrees consistent with their respective TH activities.     

Exchanges of short polymorphic DNA segments predating speciation in feline major histocompatibility complex class I genes

Sequence comparisons of 14 distinct MHC class I cDNA clones isolated from species representing the three major taxonomic lineages of Felidae (domestic cat lineage, ocelot lineage, and pantherine lineage) revealed that feline MHC class I alleles have highly mosaic structures with short polymorphic sequence motifs that are rearranged between alleles of individual MHC loci, between MHC class I genes within cat species, and between homologous MHC loci in different species. The pattern of sequence variation in felids supports the role of the following factors in production and maintenance of MHC variation: (1) gradual spontaneous mutation; (2) selective pressure to conserve certain residues but also to vary in hypervariable regions, notably residues that functionally participate in antigen recognition and presentation; and (3) recombination-mediated gene exchange between alleles and between related genes. The overall amount of genetic variation observed among MHC class I genes in the Felidae family is no greater than the amount of variation within any outbred cat species (i.e., domestic cat, ocelot). The occurrence of equivalent levels of polymorphism plus the simultaneous persistence of the same sequence motifs in divergent feline species suggest that most MHC class I nucleotide site polymorphism predated species divergences. Ancient polymorphisms have been transmitted through the speciation events and modern feline MHC class I alleles were derived by recombinational exchange of polymorphic sequence motifs. Moreover, some of these sequence motifs were found in other mammalian MHC class I genes, such as classical human HLA-B5, nonclassical human HLA-E class I genes, and bovine class I genes. These results raise the prospect of an ancient origin for some motifs, although the possibility of convergence in parallel mammalian radiations cannot be excluded. Correspondence to: N. Yuhki