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.     

昆虫抗冻蛋白的分离纯化及特性分析

昆虫抗冻蛋白具有很高的热滞活性,可保护机体免受结冰引起的伤害。昆虫抗冻蛋白的分离纯化多采用凝胶过滤层析、离子交换层析及HPLC等技术,已用于鱼类抗冻蛋白纯化的冰亲和纯化(IAP)技术也可考虑应用于昆虫抗冻蛋白的分离提纯。昆虫抗冻蛋白具有高活性,规则的一级结构及类似的冰晶结合表面等特性。     

植物抗冻蛋白研究进展

抗冻蛋白(AFPs)最初是从极区海鱼中发现的一种适应低温的特异蛋白质, 它能阻止体液内冰核的形成与生长,维持体液的非冰冻状态.对近年来植物AFPs的发现过程,AFP的生化特性,抗冻作用机制,抗冻蛋白基因工程及其应用前景等作了系统的综述.     

The role of endogenous antifreeze protein enhancers in the hemolymph thermal hysteresis activity of the beetle Dendroides canadensis

Antifreeze proteins (AFPs) lower the freezing point of water by a non-colligative mechanism, but do not lower the melting point, therefore producing a difference between the freezing and melting points termed thermal hysteresis. Thermal hysteresis activity (THA) of AFPs from overwintering larvae of the beetle Dendroides canadensis is dependent upon AFP concentration and the presence of enhancers of THA which may be either other proteins or low molecular mass enhancers. The purpose of this study was to determine the relative contributions of endogenous enhancers in winter D. canadensis hemolymph.Winter hemolymph collected over four successive winters (1997-1998 to 2000-2001) was tested. The first three of these winters were the warmest on record in this area, while December of the final year was the coldest on record. Protein and low molecular mass enhancers raised hemolymph THA 60-97% and 35-55%, respectively, based on hemolymph with peak THA for each year collected over the four successive winters. However, the hemolymph AFPs were not maximally enhanced since addition of the potent enhancer citrate (at non-physiologically high levels) resulted in large increases in THA. ¹³NMR showed that glycerol was the only low molecular mass solute present in sufficiently high concentrations in the hemolymph to function as an enhancer. Maximum THA appears to be ∼8.5 °C.     

昆虫抗冻蛋白的结构与生物学特性研究

抗冻蛋白(antifreeze proteins AFPs)是一类抑制冰晶生长的蛋白质,它能以非依数性形式降低溶液的冰点而对其熔点影响甚微,因而也被称作热滞蛋白。近几年来对于昆虫抗冻蛋白的研究取得了较快的发展,已有20多种昆虫抗冻蛋白被分离纯化。就昆虫抗冻蛋白的结构特征、生物学特性以及在农业、医学和食品工业等方面的应用进行介绍。     

Short Communication: Increased gene dosage augments antifreeze protein levels in transgenic Drosophila melanogaster

One of the principal environmental adaptations of certain fishes inhabiting polar and northern coastal waters is the synthesis of antifreeze proteins (AFPs). AFPs bind to and prevent the growth of nascent ice crystals, thus depressing the serum freezing point. The transgenic expression of AFP holds great promise for conferring freeze resistance to commercially important plant and animal species. Since fish at the greatest risk of freezing have multiple AFP gene copies in order to synthesize higher levels of this protein, we have evaluated this evolutionary strategy as a way to maximize AFP expression in a model transgenic host, the fruit fly Drosophila melanogaster. A construct in which AFP genes of the Atlantic wolffish are fused to the Drosophila yolk protein 1,2 promoter/enhancer region was transferred to flies through P-element mediated transformation. Several independent transgenic fly lines were used in genetic crosses to obtain multi-insert lines. Haemolymph freezing point depression (thermal hysteresis) was greater in homozygotes relative to heterozygotes for a given insert. Similarly, multi-insert lines consistently displayed greater haemolymph AFP activity than the single insert lines from which they were derived. The thermal hysteresis value obtained with a fly line harboring 8 AFP gene copies, 0.43 °C, represents the highest such value to date recorded in a transgenic host, and is even higher than the levels found in some AFP-producing fish.     

Molecular characterization and sequencing of antifreeze proteins from larvae of the beetle Dendroides canadensis

The deduced amino acid sequences of antifreeze proteins (AFPs) from larvae of the beetle Dendroides canadensis were determined from both complementary DNAs (cDNAs) and from peptide sequencing. These consisted of proteins with a 25-residue signal peptide and mature proteins 83 (Dendroides antifreeze protein; DAFP-1) or 84 (DAFP-2) amino acids in length which differed at only two positions. Peptide sequencing yielded sequences which overlapped exactly with those of the deduced cDNA sequences of DAFP-1 and DAFP-2, while the partial sequence of another AFP (DAFP-3) matched 21 of 28 residues. Seven 12- or 13-mer repeating units are present in these antifreeze proteins with a consensus sequence consisting of: Cys-Thr-X₃-Ser-X₅-X₆-Cys-X₈-X₉-Ala-X₁₁-Thr-X₁₃, where X₃ and X₁₁ tend toward charged residues, X₅ tends toward threonine or serine, X₆ toward asparagine or aspartate, X₉ toward asparagine or lysine, and X₁₃ toward alanine in the 13-mers. The most interesting feature of these proteins is that throughout the length of the mature antifreeze proteins every sixth residue is a cysteine. These sequences are not similar to any of the known fish AFPs, but they are similar to AFPs from the beetle Tenebrio molitor. Accepted: 14 November 1997     

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.     

Expression of Two Self-enhancing Antifreeze Proteins from the Beetle <Emphasis Type="Italic">Dendroides canadensis</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Antifreeze proteins depress the non-equilibrium freezing point of aqueous solutions, but only have a small effect on the equilibrium melting point. This difference between the freezing and melting points has been termed thermal hysteresis activity (THA). THA identifies the presence and relative activity of antifreeze proteins. Two antifreeze protein cDNAs, dafp-1 and dafp-4, encoding two self-enhancing (have a synergistic effect on THA) antifreeze proteins (DAFPs) from the beetle Dendroides canadensis, were introduced into the genome of Arabidopsis thaliana via Agrobacterium-mediated floral dip transformation. Southern blot analysis indicated multiple insertions of transgenes. Both DAFP-1 and/or DAFP-4 were expressed in transgenic A. thaliana as shown by RT-PCR and Western blot. Apoplastic fluid from T ₃ DAFP-1 + DAFP-4-producing transgenic A. thaliana exhibited THA in the range of 1.2–1.35°C (using the capillary method to determine THA), demonstrating the presence of functioning antifreeze proteins (with signal peptides for extracellular secretion). The freezing temperature of DAFP-1 + DAFP-4-producing transgenic A. thaliana was lowered by approximately 2–3°C compared with the wild type.     

Partitioning of fish and insect antifreeze proteins into ice suggests they bind with comparable affinity

Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.     

Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca(2+)-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca(2+)-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca(2+)-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.     

昆虫对低温的适应——抗冻蛋白研究进展

昆虫抗冻蛋白的研究主要在几种昆虫中展开,到目前为止已有二十多种昆虫抗冻蛋白被分离纯化。本文综述了关于昆虫抗冻蛋白的结构、组成、生物学活性及功能等方面的研究进展。昆虫抗冻蛋白的二级结构为β折叠和β转角,在其特殊的氨基酸序列结构中,半胱氨酸形成的二硫键对稳定其结构和活性起着很重要的作用。影响昆虫抗冻蛋白的因子,如活化蛋白及低分子量溶质的发现开辟了昆虫抗冻蛋白研究的新领域。     

Local ice melting by an antifreeze protein

Antifreeze proteins, AFP, impede freezing of bodily fluids and damaging of cellular tissues by low temperatures. Adsorption-inhibition mechanisms have been developed to explain their functioning. Using in silico Molecular Dynamics, we show that type I AFP can also induce melting of the local ice surface. Simulations of antifreeze-positive and antifreeze-negative mutants show a clear correlation between melting induction and antifreeze activity. The presence of local melting adds a function to type I AFPs that is unique to these proteins. It may also explain some apparently conflicting experimental results where binding to ice appears both quasipermanent and reversible.     

Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, T_m, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.     

鱼类抗冻蛋白的研究进展

抗冻蛋白 (AFP)可非依数性地降低溶液冰点 ,对冷冻细胞和胚胎具有高效的保护作用。目前的研究表明 ,不同的鱼类抗冻蛋白尽管都具有降低冰点的活性 ,但在结构和组成上又存在有较大的差异。根据其结构和化学组成 ,一般将它们分为 4大类 :AFP I、AFP II、AFP III和AFP IV。抗冻蛋白的编码基因为基因组中多拷贝基因家族的成员 ,其基因表达在很大程度上要受到季节变化的影响。目前 ,普遍使用吸附抑制假说来解释AFP非依数性降低溶液冰点的分子机制 ,但不同类抗冻蛋白在降低溶液冰点时的作用模式却不尽相同。现就鱼类的 4类抗冻蛋白的结构组成、基因性质、抗冻机制及其在细胞和胚胎冻存中的作用等领域的研究进展进行概括性综述     

昆虫抗冻蛋白的研究进展

热滞效应(Tberm Hysteresis Action)最早在昆虫研究中发现,后来研究表明,它是抗冻蛋白(Antifreeze Proteins,AFPs)的一种基本性质。和鱼类,植物AFPs相比,昆虫AFPs具有更高 的热滞活性和独特的化学结构特征。昆虫AFPs在昆虫抗冻生理过程中起着相当重要的作用,表现在以下三个方面：①抑制一些冰晶形成;②提高冰冻耐受性;③可能参与水分平衡过程。光周期,气温和湿度是调控AFPs生物合成与降解的三种外部因子,而体内激素的变化可能是直接调节脂肪体合成AFPs的内部因子。     

甲虫抗冻蛋白热滞活性的二维吸附结合模型

甲虫抗冻蛋白是一种具有规则结构的昆虫抗冻蛋白。在相同浓度条件下,甲虫抗冻蛋白比鱼类抗冻蛋白有更高的热滞活性,目前已成为人们重点研究的一类抗冻蛋白。根据甲虫抗冻蛋白的结构特点及其在冰晶表面的吸附模式,应用二维吸附结合模型计算分析了具有6￣11个β-螺旋(β-helix)结构片段的甲虫抗冻蛋白变体分子,得到了它们的热滞活性随溶液浓度变化的规律,特别是热滞活性与甲虫抗冻蛋白的β-螺旋结构片段数的关系。结果显示,抗冻蛋白在冰晶表面的覆盖度是一个影响其热滞活性的重要因素。     

Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions

Expanding cryopreservation methods to include a wider range of cell types, such as those sensitive to freezing, is needed for maintaining the viability of cell-based regenerative medicine products. Conventional cryopreservation protocols, which include use of cryoprotectants such as dimethylsulfoxide (Me₂SO), have not prevented ice-induced damage to cell and tissue matrices during freezing. A family of antifreeze proteins (AFPs) produced in the larvae of the beetle, Dendroides canadensis allow this insect to survive subzero temperatures as low as −26 °C. This study is an assessment of the effect of the four hemolymph D. canadensis AFPs (DAFPs) on the supercooling (nucleating) temperature, ice structure patterns and viability of the A10 cell line derived from the thoracic aorta of embryonic rat. Cryoprotectant solution cocktails containing combinations of DAFPs in concentrations ranging from 0 to 3 mg/mL in Unisol base mixed with 1 M Me₂SO were first evaluated by cryomicroscopy. Combining multiple DAFPs demonstrated significant supercooling point depressing activity (∼9 °C) when compared to single DAFPs and/or conventional 1 M Me₂SO control solutions. Concentrations of DAFPs as low as 1 μg/mL were sufficient to trigger this effect. In addition, significantly improved A10 smooth muscle cell viability was observed in cryopreservation experiments with low DAFP-6 and DAFP-2 concentrations in combination with Me₂SO. No significant improvement in viability was observed with either DAFP-1 or DAFP-4. Low and effective DAFP concentrations are advantageous because they minimize concerns regarding cell cytotoxicity and manufacturing cost. These findings support the potential of incorporating DAFPs in solutions used to cryopreserve cells and tissues.     

Expression of an insect (Dendroides canadensis) antifreeze protein in Arabidopsis thaliana results in a decrease in plant freezing temperature

Transgenic Arabidopsis thaliana plants which express genes encoding insect, Dendroides canadensis, antifreeze proteins (AFP) were produced by Agrobacterium-mediated transformation. The antifreeze protein genes, both with and without the signal peptide sequence (for protein secretion), were expressed in transformed plants. Thermal hysteresis activity (indicating the presence of active AFPs) was present in protein extracts from plants expressing both proteins and was also detected in leaf apoplast fluid from plants expressing AFPs with the signal peptide. Transgenic lines did not demonstrate improved ability to survive freezing when compared to wild-type. However, when cooled under four different regimes, transgenic lines with AFPs in the apoplast fluid froze at significantly lower temperatures than did wild-type, especially in the absence of extrinsic nucleation events.