Báo cáo khoa học: Fully active QAE isoform confers thermal hysteresis activity on a defective SP isoform of type III antifreeze protein docx

Fully active QAE isoform confers thermal hysteresis
activity on a defective SP isoform of type III
antifreeze protein
Manabu Takamichi
1,2
, Yoshiyuki Nishimiya
1
, Ai Miura
1
and Sakae Tsuda
1,2
1 Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and
Technology (AIST), Sapporo, Japan
2 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan
Antifreeze proteins (AFPs) function to inhibit the
growth of naturally generated hexagonal ice crystals in
supercooled water by specific accumulation onto a set
of oxygen atoms constructing specific planes of the
crystals [1]. The vacant narrow spaces on an ice plane
between the bound AFPs can undergo ice growth to
form a convex ice front, which is energetically unfavor-
able for the further incorporation of water molecules
(the ‘adsorption inhibition’ model [2–4]). The tempera-
ture at which ice growth is initiated is commonly
referred to as the hysteresis freezing point (T
f
). A tem-
perature difference between the melting point (T
m
)
and T

f
observed for an ice crystal in the presence of
AFPs has been defined as thermal hysteresis (TH) [5],
which is now generally recognized as a measure of the
potency of antifreeze activity.
AFPs have been found in various organisms, such
as fish, insects, plants, fungi and bacteria, adapted to
subzero temperature environments [1]. Of these, fish
express type I–IV AFPs and antifreeze glycoprotein
(AFGP), which are structurally diverse and form the
ice-binding surface in different ways. Significantly, all
types of AFP and AFGP are expressed as mixtures of
several isoforms [1,4], and a cooperative effect between
Keywords
cooperative effect; ice growth inhibition;
notched-fin eelpout; thermal hysteresis;
type III antifreeze protein
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, Sapporo
062-8517, Japan
Fax: +81 11 857 8983
Tel: +81 11 857 8983
E-mail:
(Received 15 November 2008, revised 29
December 2008, accepted 5 January 2009)
doi:10.1111/j.1742-4658.2009.06887.x

Type III antifreeze protein is naturally expressed as a mixture of sulfopro-
pyl-Sephadex (SP) and quaternary aminoethyl-Sephadex (QAE)-binding
isoforms, whose sequence identity is approximately 55%. We studied the
ice-binding properties of a SP isoform (nfeAFP6) and the differences from
those of a QAE isoform (nfeAFP8); both of these isoforms have been
identified from the Japanese fish Zoarces elongatus Kner. The two isoforms
possessed ice-shaping ability, such as the creation of an ice bipyramid, but
nfeAFP6 was unable to halt crystal growth and exhibited no thermal
hysteresis activity. For example, the ice growth rate for nfeAFP6 was
1000-fold higher than that for nfeAFP8 when measured for 0.1 mm protein
solution at 0.25 °C below the melting point. Nevertheless, nfeAFP6 exhib-
ited full thermal hysteresis activity in the presence of only 1% nfeAFP8
(i.e. [nfeAFP8] ⁄ [nfeAFP6] = 0.01), the effectiveness of which was indistin-
guishable from that of nfeAFP8 alone. We also observed a burst of ice
crystal growth from the tip of the ice bipyramid for both isoforms on low-
ering the temperature. These results suggest that the ice growth inhibitory
activity of an antifreeze protein isoform lacking the active component is
restored by the addition of a minute amount of the active isoform.
Abbreviations
AFGP, antifreeze glycoprotein; AFP, antifreeze protein; IGM, ice growth modifier; nfeAFP6 and 8, SP and QAE isoforms of type III AFP from
notched-fin eelpout; QAE, quaternary aminoethyl; SP, sulfopropyl; T
f,
freezing point; TH, thermal hysteresis; T
m,
melting point.
FEBS Journal 276 (2009) 1471–1479 ª 2009 The Authors Journal compilation ª 2009 FEBS 1471
isoforms with respect to the TH value has been identi-
fied [6–8]. For example, an AFGP based on a repeti-
tive polypeptide consisting of Thr–Ala–Ala tripeptide
units changes the level of T

f
depression by the addition
of peptides of other lengths [6,7]. Insect AFP from
Dendroides canadensis larva, an isoform mixture of
repetitive b-helical polypeptides, interacts among the
isoforms, which affects the observed TH value [8].
Thus, the characterization of such isoforms and their
cooperative effects on antifreeze activity will aid in our
understanding of the natural production of various
AFP isoforms.
Type III AFP is a 7 kDa globular protein that
exhibits high sequence identity with the C-terminal
domain of human sialic acid synthase [9,10]. Type III
AFP was first discovered in the ocean pout Macrozoar-
ces americanus as a mixture of 12 isoforms that can be
grouped into 11 sulfopropyl (SP)- and one quaternary
aminoethyl (QAE)-Sephadex-binding species [11]. The
SP and QAE isoforms show approximately 55%
sequence identity [12]. Immunological cross-reactivity
studies have shown a significant difference between the
two isoforms [9], and detailed structural determina-
tions by X-ray crystallography and NMR spectroscopy
have indicated that the SP and QAE isoforms con-
struct a very similar tertiary fold characterized by a
two-fold symmetric motif [13–20], which provides a
large, flat, amphipathic ice-binding surface [21–24].
Further, ice etching experiments [18,25] and computer
simulations [18,23,26] have shown that type III AFP
can undergo complementary binding to a set of oxygen
atoms located on the {10


1 0} prism plane.
We have examined the cooperative effects between
several SP and QAE isoforms of type III AFP using a
commercial freezing point osmometer [27]. This device
determines the T
f
value by automatic measurement of
the ice–water equilibrium temperature of a sample
solution. We found that the SP isoform has no appre-
ciable TH activity by itself. However, we recently
reported that a recombinant SP isoform can modify
the shape of an ice crystal into a hexagonal bipyramid
below T
m
, suggesting the possibility that the SP iso-
form itself possesses some ability to control the growth
of ice crystals, although the freezing point osmometer
was unable to detect this ability. These considerations
led us to examine the details of ice growth inhibition
by the SP isoform and the difference between its activ-
ity and that of the QAE isoform.
In this study, we observed the morphological change
in an ice crystal prepared in solutions of the SP iso-
form, the QAE isoform and their mixture employing
our custom-made photomicroscope system [28] to
evaluate the abilities of these isoforms to inhibit the
growth of ice (i.e. TH activity). We used a recombi-
nant nfeAFP6 as the SP isoform and nfeAFP8 as the
QAE isoform, both of which were identified from

Zoarces elongatus Kner [27]. We discuss the ability of
ice growth inhibition of the SP and QAE isoforms on
the basis of a detailed analysis of the morphological
change in the ice crystal.
Results
Recombinant type III AFP isoforms from notched-
fin eelpout, nfeAFP6 and nfeAFP8, were used as
the SP and QAE isoforms, respectively. The primary
sequences of the two isoforms are described in Materi-
als and methods. We first examined the ice growth
inhibitory ability of each recombinant, as well as that
of a negative control (lysozyme), using a photomicro-
scope system developed previously [28]. In the
nfeAFP8 solution, the morphology of a hexagonal ice
nucleus was modified into a bipyramidal shape, as
shown in Fig. 1 (photograph and illustration a). When
the crystal growth of this ice bipyramid was measured
at a cooling rate of 0.20 °CÆmin
)1
, the growth was
strongly inhibited at temperatures below T
m
(Fig. 1,
photograph and illustrations a–c). On further cooling,
a bursting crystal elongation (i.e. crystal growth)
occurred suddenly and rapidly from the tip of the ice
bipyramid (Fig. 1, photograph and illustration d).
Here, we define this bursting temperature from the
bipyramidal tip as T
burst

. As no significant ice growth
occurred for nfeAFP8 until T
burst
, this temperature
was identified as the ice growth initiation temperature,
or simply the hysteresis freezing point (T
f
) of this solu-
tion [28]. In the case of nfeAFP8, TH = |T
m
) T
f
|
could hence be evaluated with T
m
and T
burst
.
We also observed a bipyramidal ice crystal for
nfeAFP6 at a cooling rate of 0.20 °CÆmin
)1
(Fig. 1,
photograph and illustration e). This ice bipyramid,
however, expanded rapidly and continuously with
retention of the a-toc-axis ratio (1 : 3) (Fig. 1, photo-
graph and illustrations e–g), followed by a bursting
elongation of the ice crystal suddenly from the tip of
the ice bipyramid (Fig. 1, photograph and illustra-
tion h). Such a bursting elongation was similarly
observed for nfeAFP8 (Fig. 1, photograph d). The

only difference was that the bursting elongation of
nfeAFP6 accompanied rapid crystal expansion,
whereas that of nfeAFP8 did not. We can hence evalu-
ate, for nfeAFP6, the burst initiation temperature of
an ice crystal as T
burst
. Rapid crystal expansion was
observed even at a temperature only slightly below T
m
(i.e. T
m
) 0.05 °C), which was closely similar to the
observation for the negative control (Fig. 2C); the only
Function of SP isoform of type III AFP M. Takamichi et al.
1472 FEBS Journal 276 (2009) 1471–1479 ª 2009 The Authors Journal compilation ª 2009 FEBS
difference was in the ice crystal morphology. In other
words, the ice crystal created in nfeAFP6 and negative
control solution melted above T
m
and grew below T
m
.
That is, each solution maintained an ice–water equilib-
rium state around T
m
, for which no infinite value of
the non-equilibrium freezing point could be defined.
We therefore conclude that T
m
and T

f
are equal for
nfeAFP6 solution, and that no TH activity could be
evaluated for nfeAFP6.
Table 1 shows the growth rate of the ice bipyramid
estimated for each isoform; the a-axis length of the ice
bipyramid was used for evaluation. The growth rates
were measured at two annealing temperatures between
T
m
and T
burst
(T
m
) 0.05 °C and T
m
) 0.25 °C). As
shown in Table 1, rapid and constant rates of ice
growth were evaluated for nfeAFP6, although the rates
were slower than that for the negative control (lyso-
zyme). It should be noted that, for nfeAFP6, the ice
growth (i.e. expansion) rate was decelerated by incre-
asing the protein concentration and accelerated by
lowering the annealing temperature. The crystal
expansion was observed even at a high concentration
of nfeAFP6 (5.0 mm). We also found very slow growth
(4 · 10
)2
lmÆmin
)1

) of the ice bipyramid for diluted
nfeAFP8 solution, which agrees with a previous report
by Deluca et al. [29]. When the nfeAFP8 concentration
exceeded 0.1 mm, crystal growth was halted, making it
difficult to measure the rate at T
m
) 0.05 °C. We were
able to compare the ice growth rates for the two
isoforms for their 0.1 mm solutions at T
m
) 0.25 °C;
the observed value for nfeAFP6 was approximately
1000-fold faster than that for nfeAFP8. In addition,
the growth rate of nfeAFP8 decreased slightly with
time, with only slight crystal growth along the c-axis
(data not shown).
Figure 3A shows the ice bipyramid observed in a
1 : 1 mixture of nfeAFP6 (0.05 mm) and nfeAFP8
(0.05 mm) isoforms with a total concentration of
0.1 mm at T
m
) 0.25 °C. Crystal growth was clearly
inhibited between T
m
and T
burst
for 30 min, similar
to that in the case of nfeAFP8 only. Figure 3B shows
the growth rate of the ice bipyramid for some mix-
tures of nfeAFP6 and nfeAFP8 at T

m
) 0.05 °C, the
total concentration being adjusted to 0.1 mm. The
ice growth rate decreased dramatically from 6 to
0.1 lmÆmin
)1
by the addition of only 1% nfeAFP8
a
A
B
b
c
d
e
a
b
c
d
e
f
g
h
f g
h
Fig. 1. Morphological change in an ice
crystal observed for 0.1 m
M solutions of
nfeAFP8 (QAE isoform) and nfeAFP6
(SP isoform). (A) Photomicroscope images
for nfeAFP8 (a–d) and nfeAFP6 (e–h) were

obtained at a cooling rate of 0.20 °CÆmin
)1
.
(a, e) T = T
m
) 0.1 °C; (b, f)
T = T
m
) 0.2 °C; (c, g) T = T
burst
; (d, h)
T = T
burst
after 0.05 s. (B) Illustrations of
crystal growth observed for solutions
of nfeAFP8 (a–d) and nfeAFP6 (e–h) at
different temperatures.
M. Takamichi et al. Function of SP isoform of type III AFP
FEBS Journal 276 (2009) 1471–1479 ª 2009 The Authors Journal compilation ª 2009 FEBS 1473
(i.e. [nfeAFP8] ⁄ [nfeAFP6] = 0.01), and reached a pla-
teau (0.06 lmÆmin
)1
) at 12.5% nfeAFP8. At 25%
nfeAFP8, the growth rate was indistinguishable from
that at 100% nfeAFP8.
Figure 4A shows time-course snapshots of a busting
growth of an ice bipyramid observed in a 0.1 mm solu-
tion of the 1 : 1 mixture of the two isoforms, detected
at the temperature T
burst

. Bursting growth occurred
from the tip of the ice bipyramid, as similarly observed
for the two isoforms (Fig. 1Ad and h). We were able
to estimate the TH value for this mixture, as the ice
crystal did not expand between T
m
and T
burst
(Fig. 3A), but underwent bursting crystal growth at
T
burst
(i.e. T
burst
= T
f
). TH activities of nfeAFP6,
nfeAFP8 and their 1 : 1 mixture plotted against their
A
B
C
Fig. 2. Photomicroscope images of an ice crystal observed for
0.1 m
M solutions of nfeAFP8 and nfeAFP6 between T
m
and T
burst
.
(A) An ice bipyramid observed for nfeAFP8 at T
m
) 0.25 °C; the

crystal growth was strongly inhibited for longer than 30 min. (B)
Expansion of an ice bipyramid observed for nfeAFP6 at
T
m
) 0.25 °C. The snapshots were taken before and after 2 min of
annealing time. (C) Snapshots of 0.1 m
M solution of negative con-
trol (lysozyme) annealed slightly below its T
m
value (T
m
) 0.05 °C).
The horizontal bars and arrows indicated in the photographs repre-
sent a scale of 20 lm and the direction of the c-axis, respectively.
The c-axis is vertical to the paper for the negative control.
Table 1. Ice growth rates determined for solutions of nfeAFP6,
nfeAFP8 and the negative control (lysozyme) between T
m
and
T
burst
.
Sample
Concentration
(m
M)
Annealing
temperature
(°C)
Ice

growth rate
(lmÆmin
)1
)
nfeAFP6 (SP) 0.1 T
m
) 0.05 5.9
T
m
) 0.25 13.7
1.0 T
m
) 0.05 1.8
T
m
) 0.25 3.8
5.0 T
m
) 0.05 0.6
T
m
) 0.25 1.8
nfeAFP8 (QAE) 0.01 T
m
) 0.05 4 · 10
)2
0.1 T
m
) 0.05 –
T

m
) 0.25 1 · 10
)2
Lysozyme
(negative control)
0.1 T
m
) 0.05 150
A
B
Fig. 3. (A) Photomicroscope images of an ice crystal observed for
a 0.1 m
M solution of a 1 : 1 mixture of nfeAFP6 and nfeAFP8
annealed at T
m
) 0.25 °C. The crystal showed no significant
change for 30 min. The horizontal scale bar represents 20 lm; the
arrow indicates the direction of the c-axis, which is perpendicular to
the a-axis. (B) Crystal growth rates of an ice bipyramid determined
at T
m
) 0.05 °C for nfeAFP6 in the presence of various amounts of
nfeAFP8, with the total protein concentration adjusted to 0.1 m
M.
The x-axis indicates the percentage of nfeAFP8. For example, 25%
indicates 0.075 m
M nfeAFP6 plus 0.025 mM nfeAFP8. To evaluate
the growth rate, we measured the length of the middle of the
bipyramid along the a-axis.
Function of SP isoform of type III AFP M. Takamichi et al.

1474 FEBS Journal 276 (2009) 1471–1479 ª 2009 The Authors Journal compilation ª 2009 FEBS
concentrations are shown in Fig. 4B. Interestingly, the
TH activity of the 1 : 1 mixture was closely equivalent
to that of nfeAFP8 when plotted against the total
AFP concentration. Figure 4C plots the TH activity of
a 0.1 mm solution of the mixture against the percent-
age content of nfeAFP8. Full activity was observed in
the presence of only 1% of the QAE isoform, as sug-
gested by Fig. 3B. These results indicate that nfeAFP6
exhibited full TH activity in the presence of only a
minute amount of nfeAFP8.
Figure 5 shows the value of |T
m
) T
burst
| plotted
against the protein concentration (mm) for each
nfeAFP6 and nfeAFP8 isoform. Interestingly, the two
profiles show closely similar hyperbolic curves, which
largely overlap the whole concentration range. This
suggests that the SP and QAE isoforms possess similar
levels of growth inhibitory ability for the tip of the ice
bipyramid.
Discussion
This study reveals that the SP isoform (nfeAFP6) lacks
the ability to inhibit ice growth (Figs 2 and 3), similar
A
a b c d
B
Fig. 4. (A) Burst elongation (crystal growth) arising from the tip of an ice bipyramid in a 0.1 mM solution of a 1 : 1 mixture of nfeAFP6 and

nfeAFP8. (a) An ice bipyramid just before initiation of burst growth. (b) Initiation of the crystal burst from the tip of the ice bipyramid. (c, d)
The crystal burst rapidly proceeds and leads to complete freezing of the solution. Insets show expanded views of the growth initiation point.
Horizontal bar represents 20 lm. Arrow shows the direction of the c-axis. (B) TH activities of nfeAFP6 (filled triangles), nfeAFP8 (filled
circles) and a 1 : 1 mixture of the two isoforms (open circles) as a function of each total concentration. TH was evaluated as the difference
between T
f
and T
m
(i.e. TH = |T
m
) T
f
|). For nfeAFP6, T
f
= T
m
. For nfeAFP8 and the 1 : 1 mixture, T
burst
= T
f
. (C) TH activity of nfeAFP6 in
the presence of various proportions of nfeAFP8, with the total protein concentration adjusted to 0.1 m
M.
0
0 0.1 0.2 0.3 0.4
0.1
0.2
0.3
0.4
0.5

0.6
Concentration (mM)
| T
m
- T
burst
| (ºC)
nfeAFP6 (SP)
nfeAFP8 (QAE)
Fig. 5. Concentration dependence of |T
m
) T
burst
| for nfeAFP6
(open circles) and nfeAFP8 (filled circles). T
burst
represents the tem-
perature at which a burst of ice crystal growth occurs from the tip
of the ice bipyramid. We were unable to define TH activity, but
obtained a |T
m
) T
burst
| value for nfeAFP6. For nfeAFP8, this value
was identical to the TH activity (Fig. 4B).
M. Takamichi et al. Function of SP isoform of type III AFP
FEBS Journal 276 (2009) 1471–1479 ª 2009 The Authors Journal compilation ª 2009 FEBS 1475
to the negative control, although nfeAFP6 can specifi-
cally interact with an ice nucleus to form an ice bipyra-
mid (i.e. it has ice-shaping ability). Consequently,

nfeAFP6 exhibits no TH activity and shows an ordin-
ary ice–water equilibrium phenomenon (i.e. T
m
= T
f
)
(Fig. 2). It is interesting that, although this SP isoform
was found to be a dominant component of a purified
fish type III AFP [27], it cannot function as an AFP,
but should rather be termed an ‘ice growth modifier’
(IGM), according to the nomenclature proposed by
Harding et al. [30]. Similar observations have been
reported in studies of artificial AFP mutants [31–34].
For example, a flounder type I AFP lost 90% of its
inherent TH activity and allowed continuous growth
of ice bipyramids when Ala21 was replaced with Leu
[31]. Recombinant type I AFP from shorthorn sculpin
that lacks N-terminal blocking (denoted rSS3) as well
as that of the lysine mutant of Ala25 also failed to
inhibit the growth of ice bipyramids [32,33]. For
type II AFP from longsnout poacher, continuous
growth of an ice bipyramid was observed by mutation
of Ile58, a residue located within a planar ice-binding
surface [34]. HPLC12, a QAE isoform of type III AFP
with 94% sequence identity to nfeAFP8, permits con-
tinuous growth of an ice bipyramid that expands rap-
idly on amino acid replacement of Ala16, a residue
located at the center of the ice-binding surface [29].
It has been observed that the bipyramidal ice crystal
grows continuously with retention of the c-:a-axis

ratio at approximately 3.3 for flounder type I AFP
mutants that accumulate onto a {20

21} pyramidal
plane. Significantly, a similar continuous growth of the
ice bipyramid with retention of the c-:a-axis ratio of
approximately 3 was observed for nfeAFP6 (Fig. 2),
suggesting that nfeAFP6 binds to the pyramidal plane.
In contrast, significant ice growth along the c-axis was
identified for ordinary type III AFP and sculpin type I
AFP, which bind to the prism plane. nfeAFP8 also
permitted the growth of an ice bipyramid along the
c-axis at a low concentration (0.005 mm), suggesting
that nfeAFP8 binds to the prism plane. It should be
noted that a recent study on ice etching revealed that
type III AFP can bind to several ice planes, including
the {10

10} prism and the {20

21} pyramidal plane [18].
The preliminary X-ray structure of nfeAFP6 (data not
shown) obtained was indistinguishable from that of
ordinary type III AFP (i.e. HPLC12) [14,18]. There
is a 41% sequence difference between nfeAFP6 and
AFP8, which includes Leu19 and Val20 locating at the
edge of the putative ice-binding region. Hence, we
assume that amino acid replacements of Leu19 and
Val20 differentiate the manner of ice binding between
nfeAFP6 and nfeAFP8.

The fast and slow ice growth rates evaluated for
nfeAFP6 and nfeAFP8 reveal a difference in their growth
inhibitory function. An extremely slow growth rate was
found for 0.01 mm nfeAFP8 (4 · 10
)2
lmÆmin
)1
,
Table 1) and for the mixture of approximately
0.01 mm nfeAFP8 plus 0.09 mm nfeAFP6 (10%
nfeAFP8) (Fig. 3B). As nfeAFP6 could not halt ice
growth by itself, it may be assumed that the two iso-
forms act cooperatively for ice growth inhibition.
Indeed, TH activity of the 1 : 99 mixture containing
approximately 0.001 mm nfeAFP8 (TH = 0.33 °C)
(Fig. 4C) was higher than that of 0.05 mm nfeAFP8
alone (TH = 0.26 °C) (Fig. 4B). We examined the
1
H-
15
N heteronuclear single quantum coherence spec-
trum of
15
N-labeled nfeAFP6 in the absence and pres-
ence of 20% of non-labeled nfeAFP8, and found that
the two spectra were virtually identical (Fig. S1).
Hence, we can assume no significant protein–protein
interaction between the two isoforms, which is in good
agreement with the proposed independent ice-binding
model of AFP [35,36]. Kristiansen and Zachariassen

[4] proposed a two-step irreversible adsorption of
AFPs to the ice surface; i.e. following the initial
adsorption controlled by hydrophobic forces, perma-
nent adsorption occurs on this plane by hydrophilic
forces. This proposition may account for the weak ice
growth inhibition of nfeAFP6. That is, nfeAFP6 can
undergo initial adsorption to a pyramidal ice plane,
but fails to undergo the secondary permanent adsorp-
tion on this plane. As nfeAFP8 presumably possesses
an ability to execute the two-step irreversible adsorp-
tion to the prism plane, nfeAFP6 may irreversibly bind
to the prism plane in the presence of nfeAFP8.
In the case of nfeAFP8, we observed that crystal
bursting was initiated from the tapered tip of the ice
bipyramid at T
f
(= T
burst
) (Fig. 1Ad), implying that
the tip is the weakest point. Significantly, even
nfeAFP6 can inhibit crystal bursting from the tip
between T
m
and T
burst
(Fig. 1Ae,f), similar to nfeAFP8
(Fig. 1Aa–c). The TH activities obtained for nfeAFP8
and the 1 : 1 mixture of nfeAFP6 and nfeAFP8 were
indistinguishable (Fig. 4B). The level of depression of
T

burst
for nfeAFP6 was also indistinguishable from
that for nfeAFP8 (Fig. 5). These results suggest that
the functions of nfeAFP6 and nfeAFP8 are equivalent
with regard to the inhibition of growth from the tips
of the ice bipyramid. To our knowledge, there is little
documentation about such growth inhibition of the
bipyramidal tip by type III AFP. We offer a plausible
explanation below.
The origin of an ice bipyramid is a hexagonal ice
unit (i.e. ice nucleus) generated in supercooled water
under a pressure of 1013 hPa [37]. A scheme of transi-
Function of SP isoform of type III AFP M. Takamichi et al.
1476 FEBS Journal 276 (2009) 1471–1479 ª 2009 The Authors Journal compilation ª 2009 FEBS
tion from an ice nucleus to an ice bipyramid in the
presence of AFP (Fig. 6), which was proposed many
years ago [38], is still widely accepted with no signifi-
cant revisions. Inherently, the ice growth rate on the
six prism planes is much faster than that on the basal
plane [37,39]. When AFPs are present, they accumulate
on the six prism planes and inhibit their growth along
the a
1
-
,
a
2
- and a
3
-axes (first layer in Fig. 6), but can-

not inhibit the generation of a new ice nucleus on the
first layer, namely ice growth along the c-axis [38].
AFPs further accumulate on the prism planes grown
from the new ice nucleus and a hinge region between
the second prism and the first basal plane [14], thereby
creating a hexagonal ice plate that is smaller than the
first layer. Repeated binding of AFP to the prism
plane and the generation of a smaller ice nucleus cause
successive stacking of hexagonal ice plates on the basal
plane, leading to the formation of an ice bipyramid, as
illustrated in Fig. 6. When pyramidal planes are cre-
ated by the adsorption of AFPs, the 12 equivalent
planes construct the bipyramidal ice crystal [40].
Hence, one can imagine that the tip of the ice bipyr-
amid is the basal plane or a part of the basal plane,
which is of ultimately small size. Therefore, such an
extremely narrow space of the top plane scarcely
allows elongation of the ice crystal along the c-axis
between T
m
and T
burst
, thereby maintaining bipyrami-
dal morphology (Fig. 1). Explosive ice growth along
the c-axis may be induced by slight expansion of the
narrow top plane through ice growth towards the
other axes on lowering the temperature to T
burst
(Fig. 1). That is, an increase in isoform concentration
(Fig. 5) may contribute to the stability of the top plane

of the ice bipyramid by stabilizing its prism or pyra-
midal planes, and this increases the value of
|T
m
) T
burst
|. These suppositions do not contradict the
recent observations of Scotter et al. [41]. These authors
showed that the crystal burst always occurred from the
tip (basal plane) of the ice bipyramid for most fish
AFPs, and occurred from the prism plane for insect
hyperactive AFPs. They ascribed the former observa-
tion to no binding ability of fish AFPs to the basal
plane and the latter to the binding of hyperactive
AFPs not only to the prism but also to the basal
plane. We assume that the fully active isoform of
type III AFP strongly binds to the prism plane,
whereas a defective isoform weakly interacts with the
pyramidal plane, but they possess similar abilities to
inhibit ice growth from the tip of the ice bipyramid.
In summary, we have found that a minute amount
of the active QAE isoform of type III AFP confers
TH activity on the SP isoform, which possesses no TH
activity by itself. This may imply that the large amount
of the SP isoform contained in the body fluid makes a
significant contribution to ice growth inhibition with
the help of the active isoform, thereby enabling the
host fish to survive in seawater at subzero tempera-
tures. The present findings may further suggest that
any defective antifreeze analog (e.g. IGM) could be

used as an effective TH substance by the addition of a
minute amount of the fully active antifreeze substance.
Materials and methods
Sample preparation
Recombinant proteins of the type III AFP isoforms nfeAFP6
and nfeAFP8 were prepared as described previously [27] with
some modifications. After sonication of genetically trans-
formed Escherichia coli BL21 (DE3), a soluble fraction con-
taining a recombinant isoform was dialyzed against 50 mm
citric acid buffer (pH 2.9). Cation exchange chromatography
was then performed using an Econo-Pac High S cartridge
(Bio-Rad, Hercules, CA, USA) with a linear NaCl gradient
(0–0.5 m) with 50 mm citric acid buffer (pH 2.9). The amino
acid sequences of nfeAFP6 and nfeAFP8 are as follows:
nfeAFP6, G1ESVVATQLIPINTALTPAMMEGKVTNPS
GIPFAEMSQIVGKQVNTPVAKGQTLMPGMVKTYVP
AK66; nfeAFP8, N1QASVVANQLIPINTALTLVMMRA
EVVTPMGIPAVDIPRLVSMQ VNRAVPLGTTLMPEM
VKGYTPA65. The concentration of each purified sample
was measured by UV absorption (280 nm) using a DU 530
spectrophotometer (Beckman Coulter, Fullerton, CA, USA).
c-axis
Basal plane
Prism plane
a
1
a
2
a
3

1
st
layer
2
nd
layer
3
rd
layer
Fig. 6. Left: illustration of the proposed transition scheme from ice
nucleus to ice bipyramid based on previous ideas [38]. Right: illus-
tration of an ice bipyramid observed in a solution of type III AFP.
Detailed explanations are given in the text.
M. Takamichi et al. Function of SP isoform of type III AFP
FEBS Journal 276 (2009) 1471–1479 ª 2009 The Authors Journal compilation ª 2009 FEBS 1477
Measurement of ice growth rate and TH activity
Ice crystal morphology was observed and the crystal growth
rate was measured for solutions of nfeAFP6, nfeAFP8 and
their mixtures using a custom-made photomicroscope
system described in [28]. Detailed procedures for the evalua-
tion of TH activity are also described in [28] using a cooling
rate of 0.20°CÆmin
)1
. The sample solution was placed in a
capillary tube and frozen at approximately )30 °C; it was
then warmed by manipulation of the temperature control
device until a single ice crystal was observed ( 10–30 lm).
The observed ice crystal was annealed slightly below its T
m
,

and recorded using a Color-video 3CCD camera (Sony,
Tokyo, Japan). The annealing period was 0.5–1 h for
nfeAFP6 and 3 h for nfeAFP8. The ice growth rate was
examined in five to ten still images captured at regular inter-
vals; the length of the middle of the bipyramid along the
a-axis was used for evaluations. In all solutions tested here,
the value of T
m
was )0.3 °C. This was equivalent to that of
the buffer solution (0.1 m ammonium bicarbonate, pH 7.9).
Acknowledgements
The authors thank Dr Hidemasa Kondo for providing
them with a preliminary X-ray structure of nfeAFP6.
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Supporting information
The following supplementary material is available:
Fig. S1. 500 MHz
1
H-
15
N heteronuclear single quan-
tum coherence spectra of a recombinant
15
N-labeled
protein of the defective isoform nfeAFP6 dissolved in
water in the absence and presence of non-labeled
nfeAFP8 (temperature, 4 °C; pH 6.7; [nfeAFP6] :
[nfeAFP8] = 1 : 4; total concentration, 1 mm).
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the
corresponding author for the article.
M. Takamichi et al. Function of SP isoform of type III AFP
FEBS Journal 276 (2009) 1471–1479 ª 2009 The Authors Journal compilation ª 2009 FEBS 1479