psychrotrophicus antifreeze protein generated bipyramidal ice crystals and exhibited thermal hysteresis activity for example thermal hysteresis = 0.42C for a 0.48 mm solution similar to
Trang 1antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis
Nan Xiao1,2, Keita Suzuki1,2, Yoshiyuki Nishimiya1, Hidemasa Kondo1, Ai Miura1, Sakae Tsuda1,2 and Tamotsu Hoshino1,2
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 ecologbiochem-ical 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 eukprok-aryotes, 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 = 0.42C 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 thernor-mal hysteresis activity (approximately 2C), 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 antianti-freeze 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; Tm,melting point.
Trang 2is the noncolligative depression of the freezing point
(Tf) of a solution containing ice below its melting point
(Tm) [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
0C
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 5–6C, which is much higher
than that of fish AFPs (0.5–1C) [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 (0.01–0.1C), 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 oobasidio-mycetes
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 formaobserva-tion 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
1
2
+ +
Fig 1 A total of 23 species of fungi were tested for ice modifica-tion (antifreeze) activity Strains exhibiting ice modificamodifica-tion 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.
Trang 3crystals was observed in two ascomycetes, namely
A psychrotrophicus and P camemberti The
bipyrami-dal shape of the ice crystal formed in A
psychrotrophi-cusis 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 0.3C and 0 C, respectively Protein
expression in the culture medium of A
psychrotrophi-cuswas 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- or O-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 0C, 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
28 kDa
Positive control
AnpAFP
Digested by glycosidase.
(N) (O)
(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.
Trang 4rate of 0.05CÆmin)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 0.05CÆmin)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 0.42C, 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 1.9C and that of the wild-type TisAFP was 1.1C
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
AnpAFP
TisAFP
AnpAFP
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).
Trang 5in 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 (55CÆmin)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 basidomyascomy-cetes AFP (i.e TisAFP) has been
c
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)1temperature 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.
Trang 6identified 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
0 2 4 6 8 10 12 14
2.5
2.0
1.5
1.0
0.5
0
° C)
° C)
1.5
1.0
0
0.12 0.24 0.36 0.48
° 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.
Fig 6 Photomicroscope images showing RI activity of AFPs at )6 C All samples were dissolved in 100 m M 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)1of AnpAFP, (D) 0.1 mgÆmL)1of AnpAFP, (E) 0.01 mgÆmL)1of TisAFP, (F) 0.05 mg mL)1of TisAFP and (G) 0.1 mgÆmL)1of TisAFP.
Trang 7For 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
a Ca2+-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 30C, 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
Trang 8of 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 4C on potato dextrose agar plates (PDA; Difco) containing 0.1 mgÆmL)1of ampicillin for cul-tivation, and were then stored for 2 months at 4C 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 0C 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
Trang 9observe growth initiation or melting of ice crystals to
deter-mine the nonequilibrium Tfand Tm values, respectively In
detail, Tf 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 Tmand Tfwas 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 4C
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-Select 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 25C The culture product was
pre-pared by centrifugation (18 590 g, 30 min, 4C) 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 mm 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 DE 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 Glycans in AnpAFP were detected using a glycoprotein staining kit (GelCode; TaKaRa Bio Inc., Tokyo, Japan) after SDS⁄ PAGE Horseradish peroxidase and soybean trypsin inhibitor were used as positive and negative controls, respectively Glycans were hydrolyzed by N-glyco-sidase A and O-glycoN-glyco-sidase (Roche Diagnostics GmbH, Mannheim, Germany), under optimal conditions for each enzyme, at 37C for 24 h
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