Báo cáo khoa học: Comparison of functional properties of two fungal antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis ppt

Comparison of functional properties of two fungal antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis Nan Xiao 1,2 , Keita Suzuki 1,2 , Yoshiyuki Nishimiya 1 , Hidemasa Kondo 1 , Ai Miura 1 , Sakae Tsuda 1,2 and Tamotsu Hoshino 1,2 1 Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Toyohira-ku, Sapporo, Japan 2 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Japan Introduction Many living organisms, including fungi, have biochem- ical and ecological strategies to protect themselves from freezing. Antifreeze protein (AFP) is regarded as a singular substance in such strategies, which provides freeze tolerance in several psychrophiles, such as prok- aryotes and poikilothermic eukaryotes, in a sub-zero temperature environment [1,2]. AFPs bind to the sur- face of seed ice crystals generated in an AFP-contain- ing fluid and inhibit the growth of these crystals [3]. This inhibition causes thermal hysteresis (TH), which Keywords fungal AFP; ice growth inhibition; psychrophile; recrystallization inhibition; thermal hysteresis Correspondence S. Tsuda, Functional Protein Research Group, Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan Fax: +81 11 857 8983 Tel: +81 11 857 8983 E-mail: s.tsuda@aist.go.jp (Received 8 August 2009, revised 6 November 2009, accepted 10 November 2009) doi:10.1111/j.1742-4658.2009.07490.x Antifreeze proteins are structurally diverse polypeptides that have thermal hysteresis activity and have been discovered in many cold-adapted organ- isms. Of these, fungal antifreeze protein has been purified and partially characterized only in a species of psychrophilic basidiomycete, Typhula ishikariensis. Here we report a new fungal antifreeze protein from another psychrophile, Antarctomyces psychrotrophicus. We examined its biochemical properties and thermal hysteresis activity, and compared them with those of the T. ishikariensis antifreeze protein. The antifreeze protein from A. psychrotrophicus was purified and identified as an extracellular protein of approximately 28 kDa, which halved in size following digestion with gly- cosidase. The A. psychrotrophicus antifreeze protein generated bipyramidal ice crystals and exhibited thermal hysteresis activity (for example thermal hysteresis = 0.42 °C for a 0.48 mm solution) similar to that of fish anti- freeze proteins, while a unique rugged pattern was created on the facets of the ice bipyramid. The thermal hysteresis activity of the A. psychrotrophicus antifreeze protein was maximized under alkaline conditions, while that of the T. ishikariensis antifreeze protein was greatest under acidic conditions. The T. ishikariensis antifreeze protein exhibited a bursting ice growth nor- mal to the c-axis of the ice crystal and high thermal hysteresis activity (approximately 2 °C), as in the case of insect hyperactive antifreeze pro- teins. From these results, we speculate that the A. psychrotrophicus anti- freeze protein is very different from the T. ishikariensis antifreeze protein, and that these two psychrophiles have evolved from different genes. Abbreviations AFP, antifreeze protein; AnpAFP, AFP from Antarctomyces psychrotrophicus; ITS, internal transcribed spacer; nfeAFP, AFP from notched-fin eelpout; PDA, potato dextrose agar; PDB, potato dextrose broth; RI, recrystallization inhibition; T f, freezing point; TH, thermal hysteresis; TisAFP, AFP from Typhula ishikariensis; TisAFP8, an isoform of TisAFP exhibiting a high TH activity; T m, melting point. 394 FEBS Journal 277 (2010) 394–403 ª 2009 The Authors Journal compilation ª 2009 FEBS is the noncolligative depression of the freezing point (T f ) of a solution containing ice below its melting point (T m ) [4,5]. Within the hysteresis temperature gap, AFPs modify the ice crystal habit, in that the AFP- saturated ice crystal forms a unique shape, such as a hexagonal bipyramid [6]. Recrystallization inhibition (RI) also results from the adsorption of AFP to ice crystals [4,7]. In the RI assays the size of the ice crystal shows hardly any change at temperatures close to 0 °C. AFPs have been identified in bacteria, plants, inver- tebrates and fish, and were characterized according to their structures and TH values [1,2,8]. Fish AFPs have been grouped into five types (AFPI–IV and AFGP), and insect AFPs have been grouped into three types (right- and left-handed b-helices, and a glycine-rich repeat) [8]. Although more structural information is needed for grouping plant and bacterial AFPs, they presumably have structural variations [9–12]. Insect AFPs are termed ‘hyperactive AFPs’ because their maximal TH activity is 5–6 °C, which is much higher than that of fish AFPs (0.5–1 °C) [6]. An observation of crystal burst, which is normal to the c-axis of the ice crystal, is another characteristic of hyperactive AFP [6]. In contrast, most of the plant and bacterial AFPs exhibit very weak TH (0.01–0.1 °C), although they possess RI activity [12,13]. Fungal AFPs have been discovered in snow molds that have pathogenic activities against dormant plants under snow cover [14–17]. Snow molds include two major fungal taxa of ascomycetes and basidio- mycetes and one pseudofungal taxon of oomycetes. Among them, AFP was only identified in the basid- iomycetes Coprinus psychromorbidus [15] and Typhu- la ishikariensis (TisAFP) [16]. Hoshino et al. purified the TisAFP from the culture medium and cloned the genes. TisAFP did not exhibit any similarity in pri- mary structure with other AFPs and therefore it was considered to be a representative AFP from eukary- otic microorganisms. Kawahara et al. [18] recently reported that seven strains of ascomycetes from Ant- arctica produced extracellular substances that modify ice crystal shape, although they were not identified as AFPs. As ascomycota is the largest phylum of fungi, it may be possible to identify some species found in freezing environments that adapt by pro- ducing AFPs. In this study we performed assays of antifreeze activity against culture media of a total of 23 species of ascomycetes, and identified and purified AFP from an ascomycete collected in Antarctica (AnpAFP). We believe that comparison of the biochemical characteris- tics between AnpAFP and TisAFP will provide crucial information on the molecular diversity and the distri- bution of AFPs in fungi. Results Antifreeze activity assay against ascomycetes We performed antifreeze activity assays on 1 lL of the culture medium from each of 23 psychrotrophic asco- mycetes (Fig. 1). The assay was performed by observa- tion of the ice-shaping ability (i.e. the formation of bipyramidal or hexagonal ice crystals), which indicates adsorption of AFPs to specific ice crystal planes. As shown in Fig. 1, modification of the formation of ice Antarctomyces psychrotrophicus Aniptodera chesapeakensis Aphanoascus terreus Ascomycetes sp. Ascosphaera apis Cladosporium sp. Diatrype stigma Geomyces pannorum Geotrichum candidum Graphostroma platystoma Morchella esculent Monodictys austrina Oidiodendron echinulatum Penicillium camembertii Pseudeurotium zonatum Schizosaccharmyces japonicus Sclerotinia spp. Taphrina mume Toly pocladium cylindrosporum Trichoderma hamatum Truncatella angustata Cf.Verticillium sp. 254/HP3 Verticillium sp. olrim438 12 1 2+ + Fig. 1. A total of 23 species of fungi were tested for ice modifica- tion (antifreeze) activity. Strains exhibiting ice modification in the culture medium are indicated by ‘+’, and those that did not exhibit ice modification are indicated by ‘)’. Picture 1 is the modified ice crystal of Antarctomyces psychrotrophicus and picture 2 is that of Penicillium camemberti. N. Xiao et al. Antifreeze protein from ascomycetous fungus FEBS Journal 277 (2010) 394–403 ª 2009 The Authors Journal compilation ª 2009 FEBS 395 crystals was observed in two ascomycetes, namely A. psychrotrophicus and P. camemberti. The bipyrami- dal shape of the ice crystal formed in A. psychrotrophi- cus is typical of that observed for fish AFPs [19]. After 2 months of culture of A. psychrotrophicus and P. cam- emberti, the TH activities, measured in each culture medium, were 0.3 °C and 0 °C, respectively. Protein expression in the culture medium of A. psychrotrophi- cus was monitored, during a 10-week period of culture, using SDS ⁄ PAGE) (data not shown). Protein bands, corresponding to AnpAFP, were detected from 2 weeks of culture onwards, and the concentration of AnpAFP increased consistently with time. The former result suggests that AFP is secreted into the extracellu- lar space of A. psychrotrophicus. Biochemical properties of AnpAFP Figure 2 shows the biochemical properties of AnpAFP. SDS ⁄ PAGE (Fig. 2A) followed by silver staining showed that the molecular mass of AnpAFP is approxi- mately 28 kDa. The AnpAFP was purified from the culture medium (lane B) by successive application of anion-exchange chromatography (lane C), affinity chro- matography on hydroxyapatite (lane D) and size-exclu- sion chromatography (lane E). Significantly, MALDI- TOF ⁄ MS performed for the ‘single-band’ sample of AnpAFP revealed the presence of nearly 10 different polypeptides, of approximately 21–22 kDa (Fig. 2B). Glycoprotein analysis was then performed on the AnpAFP sample using a glycoprotein staining kit (Fig. 2C). As shown in the figure, AnpAFP stained as a single pink band (lane A). It was found that after incubation with N-orO-glycosidase, AnpAFP migrates to a position corresponding to nearly half of the original molecular mass. These data indicate that AnpAFP is a glycopeptide. Figure 3 shows the results of amino acid sequence analysis for AnpAFP, and a 20-residue sequence was determined as representing its N-terminus. This sequence showed no significant similarity to the corre- sponding sequences of known AFPs from bacteria, plants, invertebrates and fish, but showed slight similar- ity to the sequence of TisAFP (Fig. 3A). As shown, seven or eight residues of the 20 were identical, or were of the same type of amino acid; therefore, it may be assumed that these two fungal AFPs share sequence identity to some extent. We examined the amino acid composition of AnpAFP in further detail, and com- pared it with that of TisAFP; the results are detailed in Fig. 3B. In AnpAFP, the most abundant residue was Asx (17.4%); however, Asx was present at a much lower frequency (3.4%) in TisAFP. In both the AFPs, Thr was the second most abundant residue (13.6% and 15.1%). In addition, it was estimated that both AFPs had relatively low contents of Arg (1.3% and 0.9%), Met (0.8% and 0.9%) and Tyr (1.9% and 2.4%). Another significant feature of AnpAFP is the presence of 2% Cys; however, Cys was not detected in TisAFP. Ice crystal morphologies of AnpAFP and TisAFP When a seed ice crystal is formed in water with a temperature lower than 0 °C, it simply expands to attain a rounded hexagonal shape when cooled at the 31 21 45 66.2 97.4 116.25 220 A A C B BCDE 28 kDa Positive control AnpAFP Digested by glycosidase. (N) (O) ABCD (kDa) Fig. 2. (A) SDS–PAGE (12.5%) after each purification step of AnpAFP. Lane A, molecular mass marker; lane B, culture medium; lane C, sample after anion exchange chromatography (High-Q col- umn); lane D, sample after affinity chromatography (hydroxyapa- tite); lane E, sample after size exclusion chromatography (sephacryl-100). AnpAFP was purified as a single band on SDS– PAGE using these purification methods. (B) Mass spectra of puri- fied AnpAFP. (C) Glycoprotein staining: lane A, AnpAFP (28 kDa); lane B, positive control (horseradish peroxidase, 45 kDa); lane C, AnpAFP after incubation with N-glycosidase; lane D, AnpAFP after incubation with O-glycosidase. After incubation with glycosidase, AnpAFP migrated to the pink band position that is nearly half of the original molecular mass of that before incubation with glycosidase. Antifreeze protein from ascomycetous fungus N. Xiao et al. 396 FEBS Journal 277 (2010) 394–403 ª 2009 The Authors Journal compilation ª 2009 FEBS rate of 0.05 °CÆmin )1 between )0.2 and )0.3 °C, as shown in the photomicroscope images A–D of Fig. 4I. A similar, but not identical, expansion of ice crystals was observed with a 0.04 mm solution of An- pAFP and the same temperature gradient. In this case, a rugged pattern was observed at the edge of the rounded hexagonal ice crystal (Fig. 4II). In a con- centrated solution of AnpAFP (0.48 mm) (Fig. 4III), the ice crystal was modified into a bipyramidal shape that is typically observed for moderately active AFPs, such as fish type I–III AFPs [19]. The only difference is that the facets of the ice bipyramid have a rugged pattern. The ice bipyramid formed almost stably on a downward temperature gradient (Fig. 4III, A–B) and, finally, exhibited a rapid elongation along the c-axis below the nonequilibrium freezing temperature (Fig. 4III, D). Figure 4IV shows the change in the crystal shape in the presence of 0.5 mm type III AFP from notched-fin eelpout (nfeAFP) when cooled in the hysteresis gap. The rapid ice crystal elongation observed following the slight crystal growth is also typical of moderately active AFPs (Fig. 4IV, C), and was ascribed to the binding of AFP to the ice crystal surface [18]. These data indicate that AnpAFP has an antifreeze activity similar to that found in fish when compared on a weight basis. Figure 4V, A–D, are photomicroscope images of an ice crystal formed in a 0.05 mm solution of a recom- binant protein of TisAFP (TisAFP8) when cooled at 0.05 °CÆmin )1 from )0.2 to )0.3 °C. These images show the process of rapid growth of the ice crystal, which is completely different from the elongation of AnpAFP and fish AFP crystals shown in Fig. 4III and IV, respectively, but is similar to the bursting pattern observed for insect hyperactive AFPs [6]. A dendritic growth pattern is observed in Fig. 4V B–D, which implies that this ice crystal bursts explosively in six directions (i.e. ±a1–a3) normal to the c-axis. Observations of the same pattern of ice crystal burst have been well documented for hyperactive AFP from snow fleas, Hypogastrura harveyi [6] and bacterial AFP from Marinomonas primoryensis [6,20], which is indicative of the binding of these hyperactive AFPs to both pyramidal and basal planes of a seed hexago- nal ice crystal. Comparison of TH activity between AnpAFP and TisAFP8 Figure 5A shows the results of a comparison of the concentration dependence of TH activity among AnpAFP, TisAFP and fish type III AFP. It also shows the concentration dependence of TH of a recombinant TisAFP isoform (denoted TisAFP8), whose amino acid sequence was recently determined by our group. As shown, the TH activity of AnpAFP was comparable to that of fish type III AFP. A maximal TH, of approxi- mately 0.42 °C, was obtained for a 0.48 mm solution of AnpAFP. Wild-type TisAFP- showed the same TH value as AnpAFP but at less than one-tenth of the AnpAFP concentration. Notably, the TH value of the recombinant TisAFP8 that produced crystal bursting (Fig. 4V) was nearly twofold higher than that of the wild-type TisAFP. The maximum TH value of recombinant TisAFP8 was 1.9 °C and that of the wild-type TisAFP was 1.1 °C. Figure 5B shows the pH-dependence of the TH value examined for AnpAFP and TisAFP (wild type). As shown, AnpAFP exhibited increased TH activities AGLDLGAASX FGALAFEGVA AGPSAVPLGT AGNYVI LAST AGPTAVPLGT AGNYAI LAST AGPTAVPLGT AGNYAI LASA AGLDLGA ASX FGALAFEGVA 1 20 10 1 20 10 AnpAFP TisAFP AnpAFP Asx 17.4 3.4 Glx 5.0 5.4 Ser 10.8 8.8 Gly 8.6 14.1 His 2.2 0.0 Arg 1.3 0.9 Thr 13.6 15.1 Ala 9.1 16.1 Pro 3.8 4.9 Tyr 1.9 2.4 Val 7.6 7.3 Met 0.8 0.9 Cys 2.0 0.0 Ile 4.0 7.8 Leu 5.4 9.3 Phe 3.6 3.4 Lys 3.9 4.9 A.Acid AnpAFP TisAFP A B Fig. 3. (A) Alignment of the N-terminal sequences of AnpAFP with those of three TisAFP isoforms. The 10th residue from the N-termi- nus of AnpAFP (marked X) could not be conclusively identified using Edman degradation. The blue and yellow residues of AnpAFP were present in all TisAFP sequences, and the green residues were identified in some sequences. (B) Comparison of the amino acid composition of AnpAFP and TisAFP (see the text). N. Xiao et al. Antifreeze protein from ascomycetous fungus FEBS Journal 277 (2010) 394–403 ª 2009 The Authors Journal compilation ª 2009 FEBS 397 in alkaline conditions; the optimal value was obtained at pH 9.3. By contrast, TisAFP exhibited higher TH activities in acidic conditions; the optimal value was obtained at pH 5.8. RI of AnpAFP and TisAFP The ability to inhibit recrystallization was assessed using photomicroscopic observation of ice crystals in AFP solutions annealed at )6 °C for 3 h, as shown in Fig. 6. In this experiment, all samples were dissolved in 100 mm ammonium bicarbonate (pH 7.9) containing 30% (w ⁄ w) sucrose (control solution), which were immediately frozen entirely by applying a fast cooling rate (55 °CÆmin )1 ). The samples were then warmed up to )6 °C and incubated at that temperature for 3 h, which enabled us to observe the time-dependent change of the ice crystals in the sample. Figure 6A shows a photomicroscope image of the control solution before 3 h of incubation. After the 3 h incubation period, the ice crystals formed in the control showed significant growth (Fig. 6B). By contrast, the sizes of the ice crys- tals in a 0.05 mgÆmL )1 AnpAFP solution remained small (Fig. 6C); they were much smaller compared with the control (Fig. 6B). Such a tendency was further emphasized by increasing the AnpAFP concentration to 0.1 mgÆmL )1 (Fig. 6D). These data imply that AnpAFP possesses RI activity. Figure 6E–G shows the photomicroscopic observations of RI activity for TisAFP at various concentrations (0.01–0.1 mgÆmL )1 ). As shown, more effective RI activity compared with AnpAFP was indicated by the smaller size of the crys- tal, which was reduced in size with increasing concen- trations of TisAFP. To our knowledge, these are the first RI data obtained for fungal AFPs. Discussion The Fungi kingdom has two major divisions: ascomy- cetes and basidomycetes. AFP (i.e. TisAFP) has been c ABCD I II III IV V c VI Fig. 4. Photomicroscope images of an ice crystal in AFP solutions, showing initiation of rapid growth or bursting in a 0.05 °CÆmin )1 temperature gradient from )0.2 to )0.3 °C. I, ice crystal expansion without AFP. II, ice crystal modified by 0.05 m M AnpAFP. III, ice crystal burst in 0.48 m M AnpAFP. IV, ice crystal burst in moderately active fish type III AFP from Notched-fin eelpout (NfeAFP) at a concen- tration of 0.04 m M. V, ice crystal burst in 0.05 m M of the wild-type TisAFP. VI, a model of ice growth inhibition of AnpAFP. The hexagonal ice plates are thought to be stacked with rotations, which will create a rugged pattern on the facets of the ice crystal. Antifreeze protein from ascomycetous fungus N. Xiao et al. 398 FEBS Journal 277 (2010) 394–403 ª 2009 The Authors Journal compilation ª 2009 FEBS identified only in basidomycetes [2,14–16]. We searched for new fungal AFPs in the psychrophilic ascomycetes listed in Fig. 1, and found that only two ascomycete species, A. psychrotrophicus and P. camemberti, had antifreeze activities in the culture media. The A. psych- rotrophicus strains were isolated from the soils of the maritime and continental areas of Antarctica, suggest- ing a wide distribution of this fungal species in Antarc- tica. By contrast, P. camemberti is a common ascomycete distributed in cold climate regions through- out the world. These AFPs showed ice-binding activi- ties, which may contribute to the cold-adaptation capability of these psychrophilic fungi. Purified AnpAFP was identified (by SDS ⁄ PAGE) as a 28 kDa protein and was assumed to be a mixture of approximately 10 peptides according to the MALDI- TOF ⁄ MS analysis (Fig. 2). As most of the natural AFPs from fish, insects, plants and microorganisms have been reported to consist of five to 10 isoforms [12,21–25], it could be assumed that AnpAFP consists of approximately 10 isoforms. Here, AnpAFP, TisAFP and AFPIII (Fig. 5) consist of a mixture of AFP iso- forms in A. psychrotrophicus, Typhula ishikariensis and Zoarces elongatus, respectively. We attempted to deter- mine the primary sequence of the isoforms of AnpAFP, and our preliminary Edman-degradation experiments showed that at least Ala1, Gly2 and Leu5 are highly conserved between the isoforms (data not shown). pH 0 0.05 0.10 0.15 0.20 0.25 02468101214 2.5 2.0 1.5 1.0 0.5 0 TisAFP AnpAFP TH ( ° C) TH ( ° C) 1.5 1.0 0 0 0.12 0.24 0.36 0.48 TH ( ° C) 2.0 TisAFP8 TisAFP wild type type III AFP(NfeAFP) AnpAFP 0.5 A B Fig. 5. (A) TH activity of AnpAFP compared with wild-type TisAFP, recombinant TisAFP8 and fish type III AFP as a function of concen- tration (m M). The AFPs include: recombinant TisAFP isoform 8 (square); wild-type TisAFP (triangle); natural AnpAFP (diamond, dashed line); and natural type III fish AFP (circle, solid line). (B) Effect of pH on the TH activities of natural AnpAFP (filled circle, solid line) and TisAFP (open circle, dashed line). The vertical line at the left shows the TH activity of 50 l M TisAFP. The vertical line at the right shows the TH activity of AnpAFP at the same concentration. A B C D E F G Fig. 6. Photomicroscope images showing RI activity of AFPs at )6 °C. All samples were dissolved in 100 mM ammonium bicarbonate (pH 7.9) containing 30% (w ⁄ w) sucrose (control solution), which were immediately frozen entirely by applying a fast cooling rate (55 °CÆmin )1 ). The sam- ples were then warmed up to )6 °C and incubated at that temperature for 3 h. Panel A shows a photomicroscope image of the control solution before the 3 h incubation. The other panels are the images after the 3 h incubation observed for (B) the control solution, (C) 0.05 mgÆmL )1 of AnpAFP, (D) 0.1 mgÆmL )1 of AnpAFP, (E) 0.01 mgÆmL )1 of TisAFP, (F) 0.05 mg mL )1 of TisAFP and (G) 0.1 mgÆmL )1 of TisAFP. N. Xiao et al. Antifreeze protein from ascomycetous fungus FEBS Journal 277 (2010) 394–403 ª 2009 The Authors Journal compilation ª 2009 FEBS 399 For AnpAFP, glycosylation was suggested by SDS ⁄ PAGE. The size of the molecule was halved after incubation with glycosidase (Fig. 2C). AnpAFP reacted with both N- and O-glycosidases. These results suggest that half of the molecular weight of AnpAFP is glycan and that both N- and O-linked glycosylation occur in AnpAFP. If the isoforms of AnpAFP are dif- ferently glycosylated, several peaks might be obtained in the MALDI-TOF ⁄ MS spectrum. We could not obtain any information about the ice-binding ability of the glycan part of AnpAFP. Direct involvement of gly- can in ice binding has been suggested only for fish AFGP consisting of an -Ala-Thr-Ala- repeating unit that links to a disaccharide, b-d-galactosyl-(1,3)-a- N-acetyl-d-galactosamine [26]. N-linked glycosylation was also suggested for AFP from carrots [27] and for aCa 2+ -dependent species of fish type II AFP [28]. There is no involvement of glycans in ice binding of these proteins because no significant change in the ice- binding activity was detected when recombinant ver- sions of these proteins (without glycan) were analyzed. Both AnpAFP and TisAFP exhibited a high content of Thr residues (Fig. 3B). Thr is generally a key resi- due in the ice-binding ability of AFP. Insect b-helical AFP is composed of a -Thr-X-Thr- repeat motif, where the OH groups in the motif are arranged in line to bind with the ice crystal [29]. In fish AFGP, Thr is conjugated with a disaccharide that is directly involved in ice binding [26]. Participation of Thr in ice binding was also suggested for fish type I–III AFPs; TH activ- ity was diminished or lost when Thr was replaced with other amino acids [22,23,28,30–33]. We hence speculate that Thr residues are involved in the ice-binding site of AnpAFP and TisAFP. Regarding the other amino acids, the contents of Ser, Met, Val and Phe are simi- lar between AnpAFP and TisAFP, while the contents of Gly, Ala, Leu and Ile differ (Fig. 3B). It is worth noting that AnpAFP contains 2% Cys, while TisAFP does not. In SDS ⁄ PAGE with b-mercaptoethanol, a large molecular mass band (> 100 kDa, data not shown) was seen together with the normal 28 kDa band, which is ascribable to the polymerization of this peptide. This result suggests that Cys residues natively form an intramolecular disulfide bond in AnpAFP, and that they are formed between the molecules in the presence of a reductant. The concentration dependence of TH was similar between AnpAFP and AFPIII (Fig. 5). AnpAFP fur- ther showed the ability to inhibit recrystallization (Fig. 6), depending on the concentration of this pep- tide. These data suggest that AnpAFP can inhibit ice growth at a level similar to that of fish type III AFP. As A. psychrotrophic survives freezing and thawing, AnpAFP might provide freeze tolerance through the effective RI activity. From the comparison of pH dependence of TH activity between AnpAFP and TisAFP8 (Fig. 5B), it was found that the function of AnpAFP is maximized at an alkaline pH (approxi- mately pH 9), while that of TisAFP8 was greatest under acidic conditions (approximately pH 5). A plausible explanation for this result is that either the ice-binding site or the whole molecule of each protein is denatured and loses its activity at different pH ranges. A large difference was found in the content of Asx between AnpAFP (17%) and TisAFP (4%); how- ever, the content of Glx was similar (approximately 5%) (Fig. 3B). AFPIII exhibited almost the same degree of TH activity as AnpAFP in the pH range 2–13 [23]. A similar result was reported for insect hyperactive AFP [Rhagium inquisitor (Ri)AFP] [34]. It seems that AnpAFP functions across a relatively wide range of pH compared with TisAFP, although its TH activity is lower. TisAFP also lost TH activity after incubation at 30 °C, while AnpAFP did not (prelimin- ary results; data not shown). All of these results sug- gest a large difference between AnpAFP and TisAFP in their basic biochemical properties. In a dilute solution of AnpAFP (0.04 mm), a unique indistinct pattern (a rounded hexagonal shape) was observed on the edge of the seed ice crystal (Fig. 4II). The ice crystal expanded slightly, retaining its mor- phology when cooled, similar to ordinary ice crystals in the absence of AFP (Fig. 4I). In a more highly con- centrated solution of AnpAFP (0.48 mm), the crystal grew into a bipyramid, similar to that observed in the presence of fish AFPs, but differed in that it had a unique rugged pattern on its facets (Fig. 4III and VI). A plausible explanation for the AFP-induced ice bipyr- amid formation has been described, with illustrations, in Takamichi et al. [35]. Briefly, binding of AFP to the prism planes, and the generation of a smaller ice nucleus on the basal planes of a hexagonal seed ice crystal, are thought to cause the successive stacking of hexagonal ice plates on the basal plane. As a conse- quence, pyramidal planes are created by the adsorption of AFPs, and the 12 equivalent planes form the bipy- ramidal ice crystal. It is highly likely that a similar type of ice binding occurs in AnpAFP because a simi- lar ice bipyramid formed (Fig. 4III) and its TH value was comparable to that of AFPIII (Fig. 5A). We assume that an exceptional feature of ice growth inhi- bition of AnpAFP is that the hexagonal ice plates are stacked with rotations, as illustrated in Fig 4.VI. This hypothesis explains the observed indistinct pattern at the edge of the hexagonal ice crystal seed (Fig. 4II), as well as the formation of a rugged pattern on the facets Antifreeze protein from ascomycetous fungus N. Xiao et al. 400 FEBS Journal 277 (2010) 394–403 ª 2009 The Authors Journal compilation ª 2009 FEBS of the ice bipyramid (Fig. 4IV). Obviously, additional experiments and consideration will be necessary to verify this hypothesis. Nevertheless, we believe that our data and hypothesis significantly contribute to the understanding of the detailed functioning of AFP, as these observations have never been reported for any other species of AFP. In the solution of recombinant TisAFP8, a seed ice crystal maintains its size and shape upon cooling in the hysteresis gap (Fig. 4V, A–B) and undergoes a crystal burst below the nonequilibrium freezing temperature (Fig. 4V, C–D). The dendritic growth pattern suggests that the direction of the crystal burst is normal to the c- axis, which is typical of hyperactive AFPs from insects (e.g. snow flea, spruce budworm, etc.) and bacteria (M. primoryensis) [6]. These observations imply that a crystal burst always occurs from the prism plane with hyperactive AFPs and is ascribed to the binding of hyperactive AFPs to both the prism and the basal plane. For most fish AFPs, the burst occurs from the tip of the ice bipyramid (basal plane, see Fig. 4IV) because of a lack of binding of fish AFPs to the basal plane. The exceptional level of TH activity (Fig. 5A), and the strong ability to inhibit ice growth (Fig. 4V), support the exceptional antifreeze activity of TisAFP8, which is comparable to that of hyperactive insect antifreezes. It should be noted that the TH value of the TisAFP natu- ral product (i.e. isoform mixture) was approximately half of that of the TisAFP8 isoform, suggesting a very low percentage of TisAFP8 in the TisAFP sample. Ti- sAFP has no cysteines and no -Thr-X-Thr- repetitions, and therefore possesses no similarity in amino acid sequence to insect hyperactive AFPs [36]. 3D structural determinations and site-directed mutagenesis experi- ments of TisAFP8 are currently in progress, and should be helpful in revealing the mechanism of binding of this exceptionally strong ice growth inhibitor. In summary, we discovered a new fungal AFP (AnpAFP) from a psychrophile, A. psychrotrophicus. AnpAFP is an extracellular protein of 28 kDa, whose size is halved following digestion with glycosidase. AnpAFP generates bipyramidal ice crystals and exhib- its TH activity that is maximized under alkaline con- ditions, and a unique rugged pattern appeared on the facets of the ice bipyramid. AnpAFP also has the ability to inhibit recrystallization. There is similarity in the N-terminal residues between AnpAFP and another fungal AFP from a basidomycete (TisAFP). However, TisAFP uniquely exhibited a high TH value and a pattern of ice crystal bursting similar to that of the insect hyperactive AFPs. These two AFPs from basidiomycetes and ascomycetes might have evolved independently. Experimental procedures Preparations of fungal strains and media Five species of fungi were isolated from various terrestrial materials (mosses, soils and algal mats), which were collected in 1996 near Great Wall station on King George Island, South Shetland Islands, and from Zhongshan sta- tion in Larsemann Hills, Prydz Bay, East Antarctica. Fungi were also collected in 2007 near Soya coast, Lutzow-Holm Bay, East Antarctica. All samples were stored at )20 °C, and were transferred to 4 °C on potato dextrose agar plates (PDA; Difco) containing 0.1 mgÆmL )1 of ampicillin for cul- tivation, and were then stored for 2 months at 4 °C. Fungal colonies created on the surface of PDA were cultured at )1 °C in potato dextrose broth (PDB). The preliminary assay showed that A. psychrotrophicus NBRC 105511 (KS-1), NBRC 105512 (Z-23) and NBRC 105513 (Syw-1) had extracellular antifreeze activities, and these strains were chosen for further experiments. We collected the strain KS-1 from the soils near Great Wall station on King George Island, Z-23, near Zhongshan station in Larsemann Hills, and collected Syw-1 from the soils in Kizahashi- hama, Skarvsnes, Soya coast. DNA was extracted from the A. psychrotrophicus strains KS-1, Z-23 and Syw-1 using the ISOPALNT II protocol (Nippon Gene Co., Ltd., Tokyo, Japan). The internal transcribed spacer (ITS) region of genomic recombinant DNA was amplified using the primer pairs ITS1-F (5¢-CTTGGTCATTTAGAGGAAGTAA) and ITS4-B (5¢-CAGGAGACTTGTACACGGTCCAG), according to Gardens and Bruns (1993) [37], with modifications; they used KOD plus polymerase (Toyobo Co., Ltd., Tokyo, Japan) instead of Taq polymerase. The PCR product was purified using the QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany); and the amino acid sequence was determined on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, CA, USA) using the primer ITS1-F. Determination of antifreeze activity The antifreeze activity of the culture medium of each strain was examined by observation of ice crystal morphology using our photomicroscope system [38] with a Leica DMLB 100 photomicroscope (Leica Microsystems AG, Wetzlar, Germany) equipped with a Linkam LK600 temperature controller (Linkam, Surrey, UK). For measuring TH activ- ity, the purified sample of AnpAFP was dissolved in water, and that of TisAFP was dissolved in 100 mm ammonium bicarbonate (pH 7.9). The culture medium or AFP solution was momentarily frozen by lowering it to )25 °C, and then it was warmed up to almost 0 °C on the sample stage to create an ice crystal seed in the solution. This sample solution was then cooled down or warmed up slightly to N. Xiao et al. Antifreeze protein from ascomycetous fungus FEBS Journal 277 (2010) 394–403 ª 2009 The Authors Journal compilation ª 2009 FEBS 401 observe growth initiation or melting of ice crystals to deter- mine the nonequilibrium T f and T m values, respectively. In detail, T f was considered as the temperature at which ice crystal growth occurs from the bipyramidal tip for AnpAFP, and from the edges of the ice disc for TisAFP. The difference between T m and T f was determined to be the TH value. All photomicroscope images and movies were recorded using a color-video 3CCD camera (Sony, Tokyo, Japan) and a personal computer. Purification of AFP from A. psychrotrophicus (AnpAFP) A 1-L culture of A. psychrotrophicus was prepared by inoc- ulating PDB with 10 mycelial discs (5 mm in diameter). These were cut from the margin of an actively growing col- ony on a PDA plate. The culture was maintained at )1 °C for 2 months without shaking. We removed the mycelia by filtration, and the resulting culture medium (500 mL) was dialyzed against 25 mm Tris ⁄ HCl buffer (pH 8.0). The dial- ysate was applied to an Econo-pac High Q column (Bio- Rad, CA, USA) equilibrated with the same buffer. The fraction that exhibited antifreeze activity was eluted with the above buffer containing 1 m NaCl. The antifreeze active fraction was dialyzed against 10 mm phosphate buffer (pH 7.2), and the dialysate was loaded onto an Econo-pac CH-II column. The flow-through fraction was analyzed for antifreeze activity and was concentrated to 1 mL using a Microcon Ultracel YM-10 (Bedford, MA, USA). The con- centrated sample was loaded onto Sephacryl-100 gel and eluted using the same buffer (10 mm phosphate buffer, pH7.2). All steps of the purification procedure were carried out at 4 °C. Purification of TisAFP from the culture medium was per- formed as described previously [15]. cDNA encoding TisAFP8 (accession number Q76CE8 in DDBJ ⁄ EMBL ⁄ GenBank) was inserted into the chromosome of a methylo- trophic yeast (Pichia partoris) using pPICZ from the Easy- SelectÔ Pichia Expression Kit (Invitrogen Co., CA, USA). The methylotrophic yeast transformant obtained was cul- tured with methanol (buffered minimal methanol-complex medium) for 5 days at 25 °C. The culture product was pre- pared by centrifugation (18 590 g, 30 min, 4 °C) and then dialyzed against 50 mm Tris ⁄ HCl (pH 8.5) containing 0.1 mm phenylmethanesulfonyl fluoride. The dialysate was applied to a 5-mL Ni-nitrilotriacetic acid Superflow column (Qiagen GmbH) equilibrated with the same buffer, and then eluted with 100 mm imidazole at 0.2 mLÆmin )1 . The fraction was dialyzed against 10 mm acetic acid buffer (pH 3.0) containing 1 m m EDTA and 0.1 mm phen- ylmethanesulfonyl fluoride. The dialyzed protein was applied onto an Econo-pac High S column and then eluted with the same buffer containing 0.1 m NaCl. The fraction containing TisAFP was dialyzed against 100 mm ammo- nium bicarbonate and then TH activity was measured. Protein analyses of AnpAFP The molecular mass of AnpAFP was measured using SDS ⁄ PAGE and MALDI-TOF ⁄ MS (Voyager DEÔ PRO; Applied Biosystems). The N-terminal amino acid sequence of purified AnpAFP was determined using an ABI 491 Pro- tein Sequencer (Applied Biosystems). The N-terminal amino acid sequence of AnpAFP was deposited in Uni- protKB ⁄ Swiss-Prot (accession number P86268). The amino acid analyses were performed by the Center of Instrumental Analysis, Hokkaido University, Sapporo. Western blot analyses of AnpAFP were carried out using rabbit poly- clonal anti-TisAFP, which was prepared in our laboratory. Purified fish type III AFP, prepared in our laboratory, was used as the negative control for western blot analyses. 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Xiao et al. Antifreeze protein from ascomycetous fungus FEBS Journal 277 (2010) 394–403 ª 2009 The Authors Journal compilation ª 2009 FEBS 403 . Comparison of functional properties of two fungal antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis Nan. activity, and compared them with those of the T. ishikariensis antifreeze protein. The antifreeze protein from A. psychrotrophicus was purified and identified