A thaumatin-like protein from larvae of the beetle Dendroides canadensis enhances the activity of antifreeze proteins

The levels of thermal hysteresis (antifreeze activity) produced by purified antifreeze proteins (DAFPs) from the larvae of the beetle Dendroides canadensis at endogenous concentrations are lower than what are present in the hemolymph of overwintering larvae. Thermal hysteresis activity of DAFPs is dependent not only on AFP concentration but also on the presence of enhancers that may be either proteins (including other hemolymph DAFPs) or low-molecular mass enhancers such as glycerol. The purpose of this study was to identify endogenous protein enhancers using yeast two-hybrid, co-immunoprecipitation, and finally the enhancement of antifreeze activity. Here we show that a thaumatin-like protein from D. canadensis, until recently known only from plants, significantly enhances the thermal hysteresis of DAFP-1 and -2. Glycerol can further this enhancement, presumably by promoting the interaction of the DAFPs and thaumatin-like protein.     

Expression of a cystine-rich fish antifreeze in transgenicDrosophila melanogaster

We have usedDrosophila melanogaster as a model system for the transgenic expression of cystine-rich Type II antifreeze protein (AFP) from sea raven. This protein was synthesized and secreted into fly haemolymph where it migrated as a larger species (16 kDa) than the mature form of the protein (14 kDa) as judged by immunoblotting.Drosophila-produced Type II AFP demonstrated antifreeze activity both in terms of thermal hysteresis (0.13 °C) and inhibition of ice recrystallization. Recombinant AFP was purified and N-terminal sequencing revealed a 17 aa extension that began at the predicted signal peptide cleavage point. The expression of all three AFP types in transgenicDrosophila has now been achieved. We conclude that the globular Type II and Type III AFPs are better choices for antifreeze transfer to other organisms than is the more widely used linear Type I AFP.     

差示扫描量热法直接测定沙冬青抗冻蛋白的热滞效应

用差示扫描量热法直接测定了从沙冬青中提取的一种抗冻蛋白（ＡＦＰ）组分的低温热行为。结果表明,该组分的低温热行为远较文献报道的各种抗冻蛋白复杂。在降、升温过程中,在低和高温侧都给出两个放或吸热峰,两个峰表现出相互独立而又相互依存的热滞行为。低温峰的热滞活性远高于高温岭。我们认为,这种ＡＦＰ分子对水及冰晶很可能有两种不同的相互作用和影响。     

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.     

Activity of short segments of Type I antifreeze protein

In this work, we present a study on the antifreeze activity of short segments of a Type I antifreeze protein, instead of the whole protein. This approach simplifies the correlation between antifreeze protein characteristics, such as hydrophilicity/hydrophobicity, and the effect of these characteristics on the antifreeze mechanism. Three short polypeptides of Type I AFP have been synthesized. Their antifreeze activity and interactions with water and ice crystals have been analyzed by various techniques such as circular dichroism spectroscopy, X-ray diffraction, differential scanning calorimetry, and osmometry. It is shown that one short segment of Type I AFP has an antifreeze activity of about 60% of the native protein activity. In this work, we demonstrate that short segments of Type I AFPs possess nonzero thermal hysteresis and result in modifications in the growth habits and growth rates of ice. This approach enables the preparation of large quantities of short AFP segments at low cost with high antifreeze activity, and opens the possibility of developing the commercial potential of AFPs.     

Low temperature stress modulated secretome analysis and purification of antifreeze protein from Hippophae rhamnoides, a Himalayan wonder plant

Plants' distribution and productivity are adversely affected by low temperature (LT) stress. LT induced proteins were analyzed by 2-DE-nano-LC-MS/MS in shoot secretome of Hippophae rhamnoides (seabuckthorn), a Himalayan wonder shrub. Seedlings were subjected to direct freezing stress (-5 °C), cold acclimation (CA), and subzero acclimation (SZA), and extracellular proteins (ECPs) were isolated using vacuum infiltration. Approximately 245 spots were reproducibly detected in 2-DE gels of LT treated secretome, out of which 61 were LT responsive. Functional categorization of 34 upregulated proteins showed 47% signaling, redox regulated, and defense associated proteins. LT induced secretome contained thaumatin like protein and Chitinase as putative antifreeze proteins (AFPs). Phase contrast microscopy with a nanoliter osmometer showed hexagonal ice crystals with 0.13 °C thermal hysteresis (TH), and splat assay showed 1.5-fold ice recrystallization inhibition (IRI), confirming antifreeze activity in LT induced secretome. A 41 kDa polygalacturonase inhibitor protein (PGIP), purified by ice adsorption chromatography (IAC), showed hexagonal ice crystals, a TH of 0.19 °C, and 9-fold IRI activity. Deglycosylated PGIP retained its AFP activity, suggesting that glycosylation is not required for AFP activity. This is the first report of LT modulated secretome analysis and purification of AFPs from seabuckthorn. Overall, these findings provide an insight in probable LT induced signaling in the secretome.     

Characterization of a novel β-helix antifreeze protein from the desert beetle Anatolica polita

Many ectotherms organisms produce antifreeze proteins (AFPs) which inhibit the growth of ice by binding to the surface of ice crystals. In this study, a novel antifreeze protein gene from the desert beetle Anatolica polita (named as Apafp752) was expressed in a high level in Escherichia coli strain BL21 (DE3). An approximately 30 kDa fusion protein thioredoxin (Trx)-ApAFP752 was purified through Ni–NTA affinity chromatography and gel filtration chromatography. The activity of the purified fusion protein Trx-ApAFP752 was analyzed by thermal hysteresis activity (THA) and cryoprotection assay. The results suggested that Trx-ApAFP752 conferred freeze resistance on bacterium in a concentration- and time-dependent manner and the cryoprotective effect increased under alkaline conditions. Circular Dichroism (CD) spectrum analysis showed that the recombinant protein of ApAFP752 possessing β-sheet as the main structure was stable under a wide range of pH from 2.0 to 11.0 and thermal stability below 50 °C. The predicted 3D structure showed that Trx-ApAFP752 could form a β-helix structure on the antifreeze protein part, which placed most of the Thr in a regular array on one side of the protein to form a putative ice-binding surface.     

Activation of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Purified antifreeze proteins (AFPs) from the larvae of the beetle Dendroides canadensis do not produce the high levels of antifreeze activity seen in the hemolymph of overwintering larvae, even when the purified AFPs are assayed at very high concentrations. However, addition of certain proteins or agar (at concentrations sufficiently low that the gel state does not result) to the Dendroides AFP resulted in a 2–3-fold increase in activity. A 70-kDa protein with AFP-activating capabilities was purified from Dendroides larvae. Addition of this endogenous activator protein to a 4 mg·ml^-1 solution of AFP increased the activity of the AFPs to values comparable to those of the hemolymph of overwintering larvae. Data derived from a modified immunoblot technique demonstrate that the activators bind to the AFP, or vice versa. Formation of this association must allow the AFP to block ice crystal growth by binding to the surface of potential seed crystals in the normal fashion. However, because the AFP-activator complex is much larger than the AFP alone, the complex probably blocks a greater surface area of the crystal and is thus a more efficient antifreeze.Abbreviations AFP antifreeze protein - BSA bovine serum albumine - DEAE diethylaminoethyl - Ig immunoglubolin - LPIN lipoprotein ice nucleator - PIN protein ice nucleator - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - TH thermal hysteresis     

Biological Characterization of Heat-stable Antifreeze Proteins from Leaves of Ammopiptanthus mongolicus

Antifreeze protein(afp) was purified from the heat stable proteins in the leaves of Ammopiptanthus mongolicus (Maxim.) Cheng f. by two-dimensional electrophoresis-electrophoretic elution. Its molecular weight and pi are about 40 kD and 9.0 respectively, and its thermal hysteresis activity (THA) is 0.9 ℃ at 20 g/L. afp is different from other antifreeze proteins. The N-terminal 20 amino acids of afp is SDDLSFTFNKFVPCQTDILF. alp is abundant in leaves and may play an important role in the antifreeze process in A. mongolicus during the period of ovenwintering.     

A leucine-rich repeat protein of carrot that exhibits antifreeze activity

A gene encoding an antifreeze protein (AFP) was isolated from carrot (Daucus carota) using sequence information derived from the purified protein. The carrot AFP is highly similar to the polygalacturonase inhibitor protein (PGIP) family of apoplastic plant leucine-rich repeat (LRR) proteins. Expression of the AFP gene is rapidly induced by low temperatures. Furthermore, expression of the AFP gene in transgenic Arabidopsis thaliana plants leads to an accumulation of antifreeze activity. Our findings suggest that a new type of plant antifreeze protein has recently evolved from PGIPs.     

新疆准噶尔小胸鳖甲抗冻蛋白基因的克隆和抗冻活性分析

根据GenBank中已发表的昆虫抗冻蛋白基因的保守序列设计引物,利用RT-PCR技术结合3'-RACE扩增的方法,从新疆荒漠昆虫准噶尔小胸鳖甲 Microdera punctipenis dzunarica中获得了长约294 bp不含信号肽的抗冻蛋白cDNA片段,命名为MpAFP5,其全长序列为363 bp(GenBank注册号为：AY821792)。基因测序结果表明, MpAFP5-cDNA片段与加拿大拟步甲Dendroides canadensis AFP 8基因片段、黄粉甲Tenebrio molitor THP 4-9基因片段的核苷酸同源性分别达68.4%和71.8%,氨基酸序列同源性分别达70%和81%。将MpAFP5构建到原核表达载体pGEX4T-1中,重组质粒pGEX4T-1-MpAFP5在大肠杆菌BL21(DE3)中能够表达融合抗冻蛋白,SDS-PAGE分析显示融合蛋白的分子量约为37 kD,Western印迹分析证明MpAFP5在大肠杆菌中正确表达。细菌的抗冻实验结果显示准噶尔小胸鳖甲融合抗冻蛋白对细菌具有显著的抗冻保护作用,保护效果与抗冻蛋白剂量呈正相关性。     

A natural variant of type I antifreeze protein with four ice-binding repeats is a particularly potent antifreeze.

A 4.3-kDa variant of Type I antifreeze protein (AFP9) was purified from winter flounder serum by size exclusion chromatography and reversed-phase HPLC. By the criteria of mass, amino acid composition, and N-terminal sequences of tryptic peptides, this variant is the posttranslationally modified product of the previously characterized AFP gene 21a. It has 52 amino acids and contains four 11-amino acid repeats, one more than the major serum AFP components. The larger protein is completely alpha-helical at 0 degree C, with a melting temperature of 18 degrees C. It is considerably more active as an antifreeze than the three-repeat winter flounder AFP and the four-repeat yellowtail flounder AFP, both on a molar and a mg/mL basis. Several structural features of the four-repeat winter flounder AFP, including its larger size, additional ice-binding residues, and differences in ice-binding motifs might contribute to its greater activity. Its abundance in flounder serum, together with its potency as an antifreeze, suggest that AFP9 makes a significant contribution to the overall freezing point depression of the host.     

Interaction of reduced nicotinamide adenine dinucleotide with an antifreeze protein from Dendroides canadensis: mechanistic implication of antifreeze activity enhancement

Antifreeze proteins (AFPs) found in many organisms can noncolligatively lower the freezing point of water without altering the melting point. The difference between the depressed freezing point and the melting point, termed thermal hysteresis (TH), is usually a measure of the antifreeze activity of AFPs. Certain low molecular mass molecules and proteins can further enhance the antifreeze activity of AFPs. Interaction between an enhancer and arginine is known to play an important role in enhancing the antifreeze activity of an AFP from the beetle Dendroides canadensis (DAFP-1). Here, we examined the enhancement effects of several prevalent phosphate-containing coenzymes on the antifreeze activity of DAFP-1. β-Nicotinamide adenine dinucleotide (reduced) (NADH) is identified as the most efficient enhancer of DAFP-1, which increases the antifreeze activity of DAFP-1 by around 10 times. Examination of the enhancement abilities of a series of NADH analogs and various molecular fragments of NADH reveals that the modifications of nicotinamide generate a series of highly efficient enhancers, though none as effective as NADH itself, and the whole molecular structure of NADH is necessary for its highly efficient enhancement effect. We also demonstrated a 1:1 binding between DAFP-1 and NADH. The binding was characterized by high-performance liquid chromatography (HPLC) using the gel filtration method of Hummel and Dreyer. The data analysis suggests binding between DAFP-1 and NADH with a dissociation constant in the micromolar range. Interactions between DAFP-1 and NADH are discussed along with molecular mechanisms of enhancer action.     

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.     

Chemical chaperoning action of glycerol on the antifreeze protein of rainbow smelt

Rainbow smelt (Osmerus mordax) accumulate high levels of glycerol and moderate levels of trimethylamine oxide (TMAO) that lower the colligative freezing point of the serum and thereby contribute to seasonal freeze resistance. In the current study, the possibility that one or both of these compounds might also have a chaperoning role at low temperatures in smelt was investigated by studying their effects on the smelt antifreeze protein (AFP). Activity of the AFP in the presence of physiological levels of TMAO and glycerol was observed by means of ice crystal morphology and measured as thermal hysteresis. Ice crystals in AFP solutions were not visibly modified by either compound and TMAO at 25 and 50 mM had no appreciable effect on hysteresis; however, glycerol at 250 and 500 mM increased hysteresis. An equiosmolar level of NaCl was not as effective as glycerol in enhancing hysteresis, suggesting that osmolarity had little or no role. Although cross-linking experiments showed dimerization of AFP to be unchanged in the presence of glycerol, circular dichroism and intrinsic fluorescence analyses revealed enhanced protein folding. As glycerol enhances the folding and consequent activity of smelt AFP, protein chaperoning appears to be an endogenous role of glycerol in this vertebrate species.     

A theoretical model of a plant antifreeze protein from Lolium perenne.

Antifreeze proteins (AFPs), found in certain organisms enduring freezing environments, have the ability to inhibit damaging ice crystal growth. Recently, the repetitive primary sequence of the AFP of perennial ryegrass, Lolium perenne, was reported. This macromolecular antifreeze has high ice recrystallization inhibition activity but relatively low thermal hysteresis activity. We present here a theoretical three-dimensional model of this 118-residue plant protein based on a beta-roll domain with eight loops of 14-15 amino acids. The fold is supported by a conserved valine hydrophobic core and internal asparagine ladders at either end of the roll. Our model, which is the first proposed for a plant AFP, displays two putative, opposite-facing, ice-binding sites with surface complementarity to the prism face of ice. The juxtaposition of the two imperfect ice-binding surfaces suggests an explanation for the protein's inferior thermal hysteresis but superior ice recrystallization inhibition activity and activity when compared with fish and insect AFPs.     

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.     

Solution Structures,Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.     

Artificial multimers of the type III antifreeze protein. Effects on thermal hysteresis and ice crystal morphology

A variant of antifreeze protein (AFP) named RD3 from antarctic eel pout (Lycodichthys dearborni) comprises the type III AFP intramolecular dimer, which is known to exhibit a significant enhancement of thermal hysteresis when compared with the type III AFP monomer (Miura, K., Ohgiya, S., Hoshino, T, Nemoto, N., Suetake, T., Miura, A, Spyracopoulos, L., Kondo, H., and Tsuda, S. (2001) J. Biol. Chem. 276, 1304-1310). Here we genetically synthesized intramolecular dimer, trimer, and tetramer of the type III AFP, for which we utilize the genes encoding the primary sequences of the N-domain, the C-domain, and the 9-residue linker of RD3, and we examined the AFP multimerization effects on thermal hysteresis and ice crystal morphology. Significantly, (i) the thermal hysteresis increases in proportion with the size of the multimers, (ii) a larger size of the multimer exerts the maximum activity at lower concentration, (iii) every multimer changes the morphology of a single ice crystal into a unique shape that is similar but not identical to the ordinary hexagonal bipyramid, and (iv) the size of ice crystal becomes dramatically small with increasing the concentration of the multimer. The thermal hysteresis enhancement of the multimer was detected in both molar and domain bases. These results suggest that a molecule comprising the multiple AFP domains connected in tandem acquires an enhanced affinity for the ice binding.