From the Division of Structural Biology and
Biochemistry, Hospital for Sick Children, Toronto, Ontario,
M5G 1X8, Canada, the § Department of Biochemistry,
University of Toronto, Toronto, Ontario, M5G 1L5 Canada, the
Department of Laboratory Medicine and Pathobiology, University
of Toronto, Toronto, Ontario, M5G 1L5, Canada, the ** Ocean Sciences
Centre, Memorial University of Newfoundland, St. John's, Newfoundland,
A1C 5S7 Canada, and the
Department of
Biological Sciences, National University of Singapore,
Singapore, 119 260, Singapore
Received for publication, October 11, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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The antifreeze polypeptides (AFPs) are found in
several marine fish and have been grouped into four distinct
biochemical classes (type I-IV). Recently, the new subclass of
skin-type, type I AFPs that are produced intracellularly as mature
polypeptides have been identified in the winter flounder
(Pleuronectes americanus) and the shorthorn sculpin
(Myoxocephalus scorpius). This study demonstrates the
presence of skin-type AFPs in the longhorn sculpin (Myoxocephalus
octodecemspinosus), which produces type IV serum AFPs. Using
polymerase chain reaction-based methods, a clone that encoded
for a type I AFP was identified. The clone lacked a signal sequence,
indicating that the mature polypeptide is produced in the cytosol. A
recombinant protein was produced in Escherichia coli and
antifreeze activity was characterized. Four individual Ala-rich
polypeptides with antifreeze activity were isolated from the skin
tissue. One polypeptide was completely sequenced by tandem MS. This
study provides the first evidence of a fish species that produces two
different biochemical classes of antifreeze proteins (type I and type
IV), and enforces the notion that skin-type AFPs are a widespread
biological phenomenon in fish.
Many diverse species of marine fish produce antifreeze
polypeptides or antifreeze glycoproteins,
AF(G)Ps,1 as a defense
mechanism against freezing in their icy seawater habitats (1-4). The
AFPs are grouped into four biochemical classifications, types I-IV,
based on their structural features. The type I Recently, a new subclass of type I AFPs have been found in winter
flounder (Pleuronectes americanus) (10) and shorthorn sculpin (Myoxocephalus scorpius) (11). These type I AFPs are synthesized as mature polypeptides in the cytosol without the signal
and prosequences found in the secreted forms of type I AFPs, and are
referred to as skin-type AFPs as they were originally isolated and
cloned from skin tissue. The secreted forms of type I AFPs were named
liver-type AFPs to denote their primary site of production and to
distinguish them from the intracellular skin-type AFPs. The
nomenclature system presented in Low et al. (11) attempts to
differentiate between skin-type and liver-type AFPs. The winter flounder and shorthorn sculpin skin-type AFPs were named wfsAFP and
sssAFP, respectively, where the first two letters (wf and ss) denote
the fish species (winter flounder and shorthorn sculpin), and the third
position (s) denotes skin-type. Accordingly, the liver-type AFPs are
represented by an (l) at the third position as in wflAFP-6, the major
winter flounder liver-type AFP previously known as HPLC-6. The
skin-type/liver-type nomenclature scheme by itself does not specify the
biochemical classification, although all skin-type AFPs found to date
are type I AFPs. Furthermore, the skin-type AFPs were previously only
found in fish that produce type I serum (liver-type) AFPs.
The longhorn sculpin (Myoxocephalus octodecemspinosus) is a
closely related species to the shorthorn sculpin (12). It is also found
off the coast of Newfoundland, but occupies relatively deeper waters as
compared with the shorthorn sculpin and migrates offshore in the colder
season. Because of its location in deeper waters, it is less likely to
encounter environmental ice. Nonetheless, the longhorn sculpin produces
the unique type IV AFP, which has not been found in any other fish
species (5-7). The type IV AFP is relatively Gln-rich (17%), highly
helical, has a proposed four-helix bundle structure (5), and is found
in the serum with production in the liver (7). The present
investigation was undertaken to determine whether the longhorn sculpin
produces skin-type AFPs, and if present, to determine their biochemical
classification. By using RT-PCR and RACE PCR methods, a full-length
clone was isolated from the skin of longhorn sculpin that encodes for a 42-residue, Ala-rich type I AFP. The sequence lacked signal and prosequences implying an intracellular localization. A recombinant protein was produced that exhibited antifreeze activity and four individual Ala-rich polypeptides were isolated from the skin tissue that demonstrated antifreeze activity. One polypeptide, lssAFP-8, was
completely sequenced by tandem MS and found to be nearly identical to
the clone sequence. This study demonstrates that the longhorn sculpin
produces skin-type, type I AFPs and that it shares a similar defense
mechanism for cold water adaptation as the shorthorn sculpin and winter
flounder. This is to our knowledge, the first fish species identified
that produces two different biochemical classes of antifreeze proteins.
Tissue Sample Collection--
Longhorn sculpin was collected
from Conception Bay, Newfoundland, Canada. Tissues were collected on
Mar. 6, 1996, and stored at Northern Analysis--
Total RNA was isolated using
TRIzolTM reagent (Life Technologies, Gaithersburg, MD).
Ten-µg aliquots of total RNA were analyzed by standard Northern
blotting procedures using HybondTM-N nylon membrane
(Amersham Pharmacia Biotech, Baie d'Urfé, QC). Probe was
produced by digesting s3-2 (11) with PstI and
SmaI (Life Technologies) to isolate the 3'-UTR minus the
poly(A+) tail and labeled with [ RT-PCR--
One µg of total RNA from each of the specified
tissues was combined with 500 ng of the LS-RT-PCR-right primer in an
annealing reaction. Then reverse transcription reagents were added at
final concentrations of 1 × first strand buffer (Life
Technologies), 10 mM dithiothreitol, and 0.5 mM for each dNTP (Amersham Pharmacia Biotech). Reverse
transcription was performed using 200 units of Moloney murine leukemia
virus-reverse transcriptase (Life Technologies) at 42 °C. PCR was
performed using Pfu polymerase (Stratagene, La Jolla, CA).
One-tenth of the RT reaction was combined with the LS-RT-PCR-right and
-left primers at concentrations of 2.5 ng/µl, 10% (v/v) glycerol,
each dNTP at 0.1 mM, 1 × Pfu polymerase buffer (Stratagene), and 2.5 units of Pfu polymerase.
Cycling conditions were: initial hold at 94 °C for 5 min, then 30 cycles of 94 °C for 30 s, 60 °C for 1 min, and 72 °C for
1 min 45 s, followed by 10 min hold at 72 °C. Reverse
transcription and PCR were carried out in a PerkinElmer Life Sciences
2400 thermocycler. Isolated RT-PCR products were ligated into
SmaI digested pBluescript II KS+/ 5'- and 3'-RACE--
5'- and 3'-RACE reactions were performed
using the SMARTTM RACE cDNA Amplification Kit
(CLONTECH, Palo Alto, CA). One µg of total RNA
from skin tissue was used to generate 3'- and 5'-RACE-ready cDNA.
Each cDNA pool was combined with the appropriate primers and
touchdown PCR amplification was performed using AdvantageTM
2 Polymerase Mix (CLONTECH) using a PerkinElmer
2400 thermocycler. Cycling conditions were those as described by the
manufacturer (CLONTECH), and where indicated,
glycerol was added at 10% (v/v). RACE reaction products were ligated
into the pT7(Blue) cloning vector (Novagen, Madison, WI).
Southern Analysis--
Genomic DNA was isolated from liver using
DNAzolTM (Life Technologies) and 10-µg aliquots were
digested with restriction endonucleases in the presence of RNase A
(Amersham Pharmacia Biotech). Probe was produced by excising the RT-PCR
product from the cloning vector, then labeled with
[ Production of Recombinant Protein, r-lssAFP(38)--
The ORF was
PCR amplified with two primers designed with NcoI and
StyI sites using Pfu polymerase (Stratagene) with
10% (v/v) glycerol. PCR products were digested with StyI
(Life Technologies) and ligated into NcoI- and
StyI-digested placIQpar8-wfsAFP-2 (13) along with its own
stop codon.
Recombinant protein was expressed in JM105 Escherichia coli
cells in LB medium containing 100 µg/ml ampicillin by
isopropyl-1-thio- Purification of Native lssAFP--
Skin tissue was homogenized
in 0.1 M ammonium bicarbonate using a
PolytronTM homogenizer (Brinkman Instruments, Rexdale, ON),
then centrifuged at 14,000 × g, and the supernatant
was aliquoted and lyophilized. Total protein concentration was
determined using Bio-Rad Protein Assay reagent (Bio-Rad) as described
by the manufacturer using bovine serum albumin as a standard.
Immunoblots were performed using the ECLTM Western blotting
detection system (Amersham Pharmacia Biotech). The primary antibody used was an unpurified polyclonal antibody preparation against wflAFP-6
prepared in rabbit that was kindly provided by Dr. P. L. Davies
(Queen's University, Kingston, ON).
Lyophilized skin tissue protein was re-dissolved in 20 mM
Tris-HCl (pH 7.5) and injected onto a Mono STM HR 5/5 (5 mm × 50 mm) column (Amersham Pharmacia Biotech) and protein was
eluted using a step-gradient of NaCl concentration using the FPLC
system (Amersham Pharmacia Biotech). Presence of type I AFPs were
determined by immunoblotting analysis. The unbound fraction was
collected and loaded onto a Mono QTM HR 5/5 (5 mm × 50 mm) column (Amersham Pharmacia Biotech) and eluted using the same
NaCl gradient. The unbound fraction was collected, dialyzed with 4 liters of 0.1 M ammonium bicarbonate, lyophilized, then
re-dissolved in 0.1% trifluoroacetic acid. Final purification was
achieved by reverse-phase HPLC using a Bondclone 10-µm
C18 (300 mm × 7.80 mm) column (Phenomenex). Peaks
containing AFPs were identified by ice crystal morphology after
lyophilization and re-dissolving in 0.1 M ammonium bicarbonate.
Antifreeze Activity Determination--
Thermal hysteresis was
assayed in 0.1 M ammonium bicarbonate using a Clifton
Nanolitre Osmometer (Clifton Technical Physics, Hartford, NY) as
previously described (15). Concentration was determined by amino acid
analysis. All values are an average of 4 to 6 measurements with thermal
hysteresis of buffer alone subtracted and error bars represent ± 1 S.D.
Trypsin and Endoproteinase Asp-N Digestion--
Trypsin (Roche
Molecular Diagnostics, Laval, QC) was re-constituted in 25 mM ammonium bicarbonate and combined with AFP samples in
0.1 M ammonium bicarbonate in a ratio of 1:50 trypsin to
protein sample. Endoproteinase Asp-N (Roche Molecular Diagnostics) was re-constituted in H2O and protein samples for Asp-N
digestion were re-dissolved in 10 mM Tris-HCl (pH 7.5).
Negative controls consisted of enzyme and buffer alone. For
r-lssAFP(38) the Asp-N to protein sample ratio was 1:125. For lssAFP-8,
the ratio of Asp-N to protein sample used was 1:200. Digestions were
carried out at 37 °C for 11 to 14 h in a PerkinElmer 2400 thermocycler. Digested samples were de-salted using
ZipTipC18TM pipette tips (Millipore, Nepean,
ON), then dried down in a Speed Vac concentrator (Savant Instruments,
Hicksville, NY).
Mass Spectrometry and Tandem MS--
A Q-TOF tandem mass
spectrometer (Micromass Canada, Montreal, QC) was employed for all mass
spectrometric measurements. All samples were dissolved in 2-10 µl of
1:1 acetonitrile:water (plus 1% (v/v) HCOOH) or 1:1 acetonitrile:water
(plus 0.2% (v/v) acetic acid). Samples were deposited onto a nanoflow
probe tip and analyzed in the nanoelectrospray mode. Quadrupole RF
settings were between 0.5 and 1.0 for MS, and 0.25 for MS/MS. The
scanning region was between 50 and 3500 with a scan time of 1 s
and a dwell time of 0.1 s. The data was analyzed using MassLynx
(Micromass Canada). Molecular weight determination was performed using
maximum entropy based procedure (MaxEnt 1). Peptide fragmentation
(MS/MS) data was first analyzed using a MaxEnt 3 procedure followed by
manual sequence calling using PepSeq.
DNA Sequencing and Amino Acid Analysis--
All DNA sequencing
was performed using the T7 SequencingTM Kit as described by
the manufacturer (Amersham Pharmacia Biotech), or by the DNA sequencing
facility of the Centre for Applied Genomics (Hospital for Sick
Children, Toronto, ON). Amino acid analysis was performed by the
Advanced Protein Technology Centre (Hospital for Sick Children).
Northern Blot Analysis--
Total RNA from tissues of a longhorn
sculpin were probed in Northern analysis using the 3'-UTR of the
shorthorn sculpin skin-type AFP (sssAFP-2) clone, s3-2 (11). Of the
tissues examined, strong specific bands were found only in the skin,
dorsal fin, and gill filaments at ~1 kilobase in size (Fig.
1A). A much weaker signal can
be detected in the stomach.
Cloning of lssAFP--
Primers were designed (LS-RT-PCR-right and
-left, see Fig. 2) from the sssAFP-2 ORF
and RT-PCR was performed on longhorn sculpin total RNA samples. A
388-bp product was detected in skin and gill filaments, but no product
was found in the liver (data not shown). One independent clone of the
RT-PCR products from each of the gill filaments and skin were sequenced
and found to be identical. Analysis of the RT-PCR product revealed a
129-bp ORF that encodes for a 42-residue, Ala-rich polypeptide (see
Fig. 2). Due to the high Ala content, the predicted
RACE PCR primers (see Fig. 2) were used in 3'- and 5'-RACE reactions on
total RNA from skin tissue. Initially, the 5'-RACE reaction produced a
smeared product, with the majority at ~150 bp in length (data not
shown). The 5'-RACE reactions were repeated with 10% glycerol (v/v)
and a PCR product of 271 bp was identified (data not shown). Three
independent clones of this product were sequenced and found to be
identical (see Fig. 2) with no start codons (ATG) upstream of the
ORF.
The 3'-RACE reaction produced four products (data not shown). Upon
partial sequencing of the four products, only the one with the
strongest signal, 3'BAND-3 showed any relation to the RT-PCR product.
Two independent clones of 3'BAND-3 where fully sequenced and found to
be identical each other. However, the two clones contained a 33-bp
deletion just after the LS-RACE-left primer when compared with the
RT-PCR sequence (data not shown). Using the 3'-BAND-3 sequence, a third
primer was designed from the sequence in front of the
poly(A+) tail, LS-RACE-right-3 (see Fig. 2), and PCR was
repeated using this primer paired with the LS-RACE-left primer in 10%
glycerol (v/v). Under these conditions, a PCR product ~30-40 bp
larger was produced and was named 3'BAND-3-right 3. Sequencing of two independent clones of 3'BAND-3-right 3 revealed that it contained the
missing 33 bp (see Fig. 2). The full-length sequence of the lssAFP (~800 bp) gene is shown in Fig. 2
(GenBankTM accession number AF306348).
To confirm the results of the Northern analysis, RT-PCR was performed
on a wider range of tissues (Fig. 1B). A qualitative examination of the RT-PCR products shows three different levels of
positive signals. The strongest positives can be found in the gill
filament, dorsal fin, and skin. Moderate signals are found in the
stomach, kidney, and intestine, with lowest signal levels found in the
brain and liver. The levels found in the brain and liver are about
10-fold lower than those found in the digestive tract tissues. As well,
Southern blotting analysis using the RT-PCR product as a probe,
indicates that the lssAFPs are encoded for by a multigene family (Fig.
3), with at least five bands that can be
distinguished in the HindIII digest of genomic DNA (Fig. 3,
lane 3).
Production of the Recombinant Protein, r-lssAFP(38)--
To assess
the functionality of the isolated ORF, a recombinant protein was
produced in E. coli as a secreted and mature polypeptide. The HPLC profile for the final purification step is shown in Fig. 4A. The results of amino acid
analysis on the recombinant protein are shown in Table
I and a molecular mass of 3452 Da was
determined by nanospray ESI-MS which was less than the predicted
monoisotopic mass of 3794 Da for the complete ORF. Furthermore, the
amino acid analysis results, while similar, were not an exact match
with the predicted amino acid composition (see Table I). Trypsin and Asp-N peptide mapping in conjunction with tandem MS sequencing covering
the entire polypeptide demonstrated that the smaller size of the
recombinant protein was the result of a C-terminal truncation (data not
shown). The complete sequence of r-lssAFP(38) consists of the first 38 residues of the lssAFP (clone) sequence (see Fig. 6), hence the name
r-lssAFP(38).
Thermal Hysteresis Activity of r-lssAFP(38)--
The activity of
r-lssAFP(38) was assayed in comparison with reverse-phase HPLC-purified
recombinant wfsAFP-2 that was produced using the unmodified expression
vector (13). Ice crystal morphology in the presence of r-lssAFP(38) is
the typical bipyramidal form observed with all AFPs (Fig. 4A,
inset). The activity curve for r-lssAFP(38) (Fig. 4B)
shows the typical hyperbolic curve that approaches a maximum and its
activity is ~13-14% higher than that of wfsAFP-2.
Tissue Distribution of Native Polypeptides--
A polyclonal
antibody preparation against wflAFP-6, anti-type I AFP, was tested for
cross-reactivity to longhorn sculpin skin proteins. This antibody
preparation is known to cross-react with all type I AFPs found in the
shorthorn sculpin and winter
flounder.2 The anti-type I
AFP polyclonal antibody preparation cross-reacted specifically with
skin protein that was approximately the same size as r-lssAFP(38) (data
not shown). Examination of a broad range of tissues using anti-type I
AFP shows that while mRNA can be detected in all tissues, native
lssAFP polypeptides are only strongly detected in the skin, dorsal fin,
and gill filaments (Fig. 1C, lanes 1, 8, and
9) and weakly detected in the stomach (Fig. 1C, lane
4).
Purification of Native Longhorn Sculpin Skin-type AFPs--
Native
lssAFPs were purified from skin tissue by cation exchange, followed by
anion exchange chromatography, with antifreeze activity detected in the
unbound fraction in both instances (data not shown). Final purification
was performed using C18 reverse-phase HPLC (Fig.
5A). The presence of active
AFPs was determined by examining the ice crystal morphology. The
bipyramidal ice crystal morphology was only detected in peaks 7-10
(data not shown). These four peaks were designated as lssAFP-7 to -10 for longhorn sculpin skin-type
AFP-7 to 10. The amino acid composition of each peak is
shown in Table I.
Aliquots of each lssAFP sample were analyzed by nanospray ESI-MS. The
molecular mass for each lssAFP is given in Table I. Two polypeptides,
lssAFP-7 and -8, were subjected to Q-TOF tandem MS sequencing after
endoproteinase digestion with trypsin, and lssAFP-8 was further
characterized by Asp-N digestion. Using the overlapping sequences
obtained from trypsin and Asp-N analysis, complete sequence coverage
was obtained for lssAFP-8 (Fig. 5B). Peptide mapping
followed by tandem MS analysis of lssAFP-8 revealed contaminating
peptides (data not shown) that would account for the discrepancies
between the determined sequence and amino acid analysis results for
lssAFP-8. Nevertheless, the sequence of lssAFP-8 is identical to the
clone sequence except for the lack of a C-terminal Lys residue. A
residual mass of 42 Da at the N-terminal end of the MH+ of
673.34 Da indicates that the polypeptide was blocked with an acetyl
group at the N terminus. The determined sequence has a predicted
monoisotopic mass of 3707.96 Da, which agrees well with the major
monoisotopic peak of 3708 Da observed in the undigested sample. Tryptic
analysis and tandem MS of lssAFP-7 clearly shows that it is also
related to the cloned lssAFP gene sequence (Fig. 5B).
However, due to lack of sample, Asp-N analysis of lssAFP-7 was not
performed, thus the sequential order of the tryptic peptides could
not be determined.
Based on the cDNA sequence of the shorthorn sculpin skin-type
AFP (sssAFP-2), the homologous skin-type AFP of the longhorn sculpin
has been cloned by RT-PCR and RACE methods. Longhorn sculpin skin-type
AFP corresponding mRNA was detected in all tissues examined including the liver by RT-PCR. Protein was only detected in the skin,
gill filaments, dorsal fin, and stomach possibly indicating regulation
at the level of translation. However, it is also possible that the more
sensitive RT-PCR method is detecting basal levels of transcription. In
the immunoblot, much stronger signals are found in tissue samples than
in r-lssAFP(38) samples. This may reflect a higher cross-reactivity of
the antibody preparation with native lssAFP variants bearing different
epitopes than those found on r-lssAFP(38). Each individual detection
method presented in Fig. 1 does not include internal controls, thus no
quantitative analysis of expression levels can be made. However, three
independent methods (Northern, RT-PCR, and immunoblotting) display
similar trends in that higher levels of lssAFPs are detected in skin, dorsal fin, and gill filaments, leading to the qualitative statement that lssAFPs are enriched in peripheral tissues.
The shorthorn sculpin and longhorn sculpin produce quite contrasting
skin-type AFPs in terms of length and sequence (see Fig. 6) even though their respective clones
are nearly identical within the UTRs (see Fig.
7). A recombinant protein, r-lssAFP(38),
was produced from the cloned sequence and it exhibited the typical ice
crystal morphology and had comparable thermal hysteresis with another
skin-type AFP. Sequencing of the expression vector indicated that the
complete ORF was present, thus the lack of the C-terminal residues may
be due to a post-translational modification. Four polypeptides with
high Ala content and strong thermal hysteresis activity were isolated
from skin tissue. The lack of the C-terminal Lys residue and removal of
the N-terminal -NH
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-helical AFPs are
found in righteye flounders and sculpins; type II lectin-like AFPs in
sea raven and herring; type III globular AFPs in eelpouts (1, 2); and
the type IV helix bundle in the longhorn sculpin (5-7). Antifreeze
proteins act by lowering the equilibrium freezing point of a solution
in a noncolligative manner (8, 9). Most of the characterized antifreeze
proteins were isolated from the serum and are produced in the liver.
Furthermore, the traditional physiological site of AFP action has been
viewed to be the serum and extracellular compartments (1, 3).
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70 °C before use.
-32P]dCTP
(Amersham Pharmacia Biotech). The nylon membrane was prehybridized in
40% formamide, 5% dextran sulfate, 1% SDS, 4 × SSC, 7 mM Tris-HCl (pH 7.5), 1 × Denhart's buffer, and 100 µg/ml denatured calf thymus DNA for 3 h at 47 °C. Probe was
added at 0.5 × 106 cpm/ml and hybridization was
performed at 48 °C for 24 h. The membrane was washed in
solutions ranging from 1 × SSC, 1% SDS to 0.1 × SSC, 0.1% SDS at 48 °C to 72 °C and autoradiography was performed.
(MBI Fermentas Inc.,
Flamborough, ON).
-32P]dCTP (Amersham Pharmacia Biotech). The membrane
was prehybridized for 8 h at 50 °C in a buffer of 50%
formamide, 600 mM NaCl, 40 mM sodium phosphate
(pH 7.4), 4 mM EDTA (pH 8.0), 1% SDS, 5 × Denhart's
buffer, and 100 µg/ml denatured calf thymus DNA. Hybridization was
performed in a fresh buffer of 50% formamide, 600 mM NaCl, 40 mM sodium phosphate (pH 7.4), 4 mM EDTA (pH
8.0), 1% SDS, and 5% dextran sulfate at 42 °C for 48 h with
probe at a concentration of 1.0 × 106 cpm/ml.
Membrane washing and autoradiography was performed as described for
Northern analysis.
-D-galactopyranoside induction as
described in Refs. 13 and 14 with minor modifications. Growth and
expression were performed at 30 °C for 18 h after induction. Protein was precipitated from the media with 100%
(NH
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Fig. 1.
Tissue distribution of longhorn sculpin
skin-type AFPs. A, Northern analysis of longhorn
sculpin tissues probed with the 3'-UTR (minus the poly(A+)
tail) of the shorthorn sculpin skin-type AFP clone, s3-2 (11). Each
lane contains 10 µg of total RNA. B, RT-PCR analysis of
longhorn sculpin total RNA from various tissues. Lanes 1-9,
tissue samples as indicated above A. Lanes 10 and
12, RT and PCR negative controls (H2O in place
of RNA sample). Lane 11, PCR positive control (RT-PCR
product in plasmid DNA). C, tissue distribution of native
lssAFPs as identified by immunoblotting with anti-type I AFP. Each lane
contains ~5 µg of total protein. Lanes 10-13, 0.5, 0.75, 1.0, and 1.5 µg of r-lssAFP(38).
-helix secondary
structure, and the apparent 11-residue repeat structure (see below),
the polypeptide encoded for by the ORF was classified as a type I AFP.
The polypeptide sequence was designated as lssAFP (for
longhorn sculpin skin-type AFP) as per the nomenclature scheme presented in Low
et al. (11) and was classified as a skin-type AFP due to its
presence in the skin tissue and the lack of a signal sequence.
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Fig. 2.
Full-length sequence of lssAFP clone.
The ORF is in capitalized text. Translation of the ORF is given
below the nucleotide sequence. PCR primer sequences are
shown above (5' 3'), or below (3'
5') the
sequence. Primer names are given in brackets before or
after their respective primers. Mismatched nucleotide
positions within primers are indicated by underlined and
italicized text.
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Fig. 3.
The lssAFPs are encoded by a multigene
family. Southern analysis of longhorn sculpin genomic DNA isolated
from liver probed with the RT-PCR product. Each lane contains 10 µg
of genomic DNA treated with restriction enzyme as indicated.
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Fig. 4.
Production of active recombinant AFP from the
lssAFP clone. A, final purification step for
recombinant longhorn sculpin skin-type AFP, r-lssAFP(38).
C4 reverse-phase HPLC profile, 20-45% acetonitrile in
0.1% trifluoroacetic acid (60 min), the r-lssAFP(38) peak is indicated
by an asterisk (*). Inset displays the ice
crystal morphology in the presence of 4.65 mM r-lssAFP(38)
in 0.1 M ammonium bicarbonate. B, activity curve
of r-lssAFP(38) compared with wfsAFP-2 (13). See Fig. 6 and text for
sequences of r-lssAFP(38) and wfsAFP-2.
Amino acid analysis results for r-lssAFP(38) and lssAFP-7 to -10
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Fig. 5.
Purification and amino acid sequence of
native lssAFP. A, C18 reverse-phase HPLC
profile of ion exchange unbound fraction (see text) in an acetonitrile
gradient of 25-45% in 0.1% trifluoroacetic acid (60 min). Peaks
analyzed are identified by numbers above each peak.
B, summary of tandem MS results for lssAFP-8 and lssAFP-7.
Peptide masses (Da) generated by Asp-N or trypsin digestion and their
corresponding sequences as determined by tandem MS are presented along
with the theoretical masses for the sequence. For lssAFP-8, the overlap
of the peptide sequences is presented. Sequences covered by tryptic
peptides are underlined and sequence covered by Asp-N digest
is highlighted in gray. The asterisk (*)
indicates nonspecific cleavages.
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Fig. 6.
Sequence alignment of type I AFPs.
Identical residues among the compared sequences are highlighted in
gray. Above the sequence alignment is the traditional
11-residue motif definition using Asn at the fourth position although
this position may also be Asp. Below that is the new motif definition
proposed here. The first and fourth positions of the two core repeats
are indicated at the top of the alignment. The conserved
MDAPA N-terminal sequence is shown in bold text (see text).
LssAFP, longhorn sculpin skin-type AFP; sslAFP-3,
shorthorn sculpin liver-type AFP (17); aslAFP-1, arctic
sculpin liver-type AFP (19); gslAFP-5, grubby sculpin
liver-type AFP (18); wfsAFP-1 to -3, winter flounder
skin-type AFPs (10); and wflAFP-6, winter flounder
liver-type AFP (HPLC-6). The polypeptide sequences for shorthorn
sculpin skin-type AFP, sssAFP-2 (11), and longhorn sculpin liver-type
AFP, lslAFP-12 (LS-12) (5) are shown below, although no
attempt was made to align these sequences with the type I AFP sequences
shown above.
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Fig. 7.
Comparison of the longhorn sculpin and
shorthorn sculpin skin-type AFP gene sequences. Alignment of the
lssAFP sequence with the shorthorn sculpin skin-type AFP clone s3-2
(11). Identical nucleotides are highlighted in gray with
white text. ORFs for both sequences are
capitalized. No alignment was performed over the ORFs.
Surprisingly, the lssAFP sequences show highest similarity to minor serum AFPs found in other sculpins (16-18), and not to sssAFP-2 (11). A comparison of the lssAFP-8 sequence, along with the translated 129-bp ORF of the clone sequence with other type I AFPs is shown in Fig. 6. The 5-residue sequence, MDAPA, can be found in the N-terminal portion of type I AFPs from four different species of fish, and in both skin-type and serum AFPs (Fig. 6). Furthermore, a large, positively charged residue (Lys or Arg) occupies the sixth position in three of the species. This level of conservation may indicate the importance of the N-terminal portion of type I AFPs for either function or structural stability. The N-terminal region of wflAFP-6 has been proposed to be an important feature for maintaining the high helical content of the polypeptide as this region possesses an elaborate hydrogen-bonding network that "caps" the N-terminal (19). The role of the MDAPA sequence in skin-type AFPs and the minor sculpin serum AFPs is currently under investigation.
Typical type I AFPs contain an 11-residue repeat motif that has been
defined as
Thr-X2-(Asp/Asn)-X7
(where X is any amino acid but usually Ala) (see Fig. 6).
The lssAFP sequence consists of two complete 11-residue repeats (core
repeats) and one "incomplete repeat" at the C-terminal. Incomplete
repeats have an Ala residue at the fourth position, similar to the
repeats found in the wfsAFPs (10). In the wfsAFPs, positions 6 and 10 of the first core repeat are always Lys and Glu which form a salt
bridge (i, i + 4) to stabilize the helix structure (KE salt
bridge). This salt bridge is conserved in all the wfsAFPs and in
wflAFP-6 where it has been visualized by x-ray crystallography (19,
20). In sculpin AFPs that contain the repeat motif, the fourth position
of the repeat is Asp and the residue immediately preceding the Thr
(position 1) is always Lys (see Fig. 6). These residues may also be
involved in salt bridge formation (KD salt bridge). Furthermore, type I AFPs that possess the putative KD salt bridge lack the KE salt bridge.
Several studies have determined that the regular arrangement of Thr
residues (due to the repeating primary sequence) is critical for
antifreeze activity and that Thr participates directly in ice-binding
(reviewed in Ref. 21). The two putative KD salt bridges in sculpin AFPs
would be in a different orientation with respect to the Thr residues
(and hence the ice-binding surface) than the KE salt bridges of the
flounder AFPs. As well, both the Lys and Asp residues were proposed to
form an ice-binding motif (19), and the specific arrangement of Thr and
Asn/Asp residues was proposed to be critical for type I AFP activity
(22, 23). However, the introduction of this ice-binding motif into a
wfsAFP background sequence actually resulted in reduced antifreeze
activity (14) and recent work (see below) has suggested that the
ice-binding face may be located on the opposite side of the Thr
residues from the putative KD salt bridges. Thus, it is quite plausible
that the high conservation of the Lys and Asp at positions 1 and 4 of
the repeat motifs in the lssAFPs and in the minor sculpin serum AFPs is
due to structural stability and not to ice binding activity.
The activity of r-lssAFP(38) is only slightly greater (~13-14%) than that of the wfsAFP-2. The minor sculpin serum AFPs (gslAFP-5 and sslAFP-3) also show similar activity levels as the skin-type AFPs (17, 18), and are significantly lower in activity when compared with the major serum AFPs (liver-type). Recently Baardsnes et al. (24) have proposed a new ice-binding face for type I AFPs composed of Thr13, Ala17, and Ala21 of wflAFP-6 based on steric mutations in this region of the helix. These three residues are also conserved in both core repeats of the lssAFPs and in the minor sculpin serum AFPs. If these conserved residues represent the ice-binding surface, then the Lys and Asp residues are not involved in ice binding and would be free to form the KD salt bridges. Taken together, these results may indicate that the traditional definition of the 11-residue repeat may be inadvertently overstating the importance of the fourth position, i.e. the Asp/Asn residue, in antifreeze activity and ice binding. In those type I AFPs where Asp is found, the key role of the Asp may be in salt bridge formation and hence stabilization of the helix structure. In addition, as these AFPs lack the KE salt bridge, the KD salt bridges may be necessary to enhance the helix structure, as a link between helix content and antifreeze activity has been demonstrated in the past (25). A more appropriate definition of the 11-residue repeat motif may be TaaXAXXAAXX (see Fig. 6). In this nomenclature, the Thr and Ala residues at positions 5, 8, and 9 are conserved in all type I AFPs, while the two Ala residues at positions 2 and 3 are almost always Ala with only a few exceptions (hence the lowercase letters). This motif definition stresses evolutionarily conserved residues, and under the assumption that the conservation is due to functionality, the key residues in ice binding.
The closer identity of the lssAFPs to the serum AFPs of other sculpins than to the shorthorn sculpin skin-type AFP is surprising due to the high identity between the longhorn and shorthorn gene sequences in the UTRs (see Fig. 7). The maintenance of UTR sequences may indicate that key translational regulatory elements exist within the 3'- and 5'-UTRs or that these regions are necessary for mRNA stability (26, 27). The identity of the UTRs indicates that the longhorn sculpin and shorthorn sculpin skin-type AFP genes shared a common ancestor gene from before the divergence of the two species. Moreover, the fact that the two species possess different serum AFPs (type I in shorthorn sculpin versus type IV in longhorn sculpin) may indicate that the circulating, or extracellular AFP, is a cold adaptation that occurred after the separation of the two species, and that liver-type and skin-type AFPs may not necessarily be evolutionarily related. However, it is also possible that the longhorn sculpin at one time also produced a serum type I AFP. A study of the sea raven, Hemitripterus americanus, has been initiated to determine whether it also possesses a homologous skin-type AFP gene. The sea raven is also of the Cottidae family (12), but produces the type II AFP as its serum AFP. With the finding that the longhorn sculpin produces two different biochemical classes of AFPs (type IV liver-type and type I skin-type), an examination of sea raven for skin-type AFPs may assist in clarifying the evolutionary relationship between the skin-type and liver-type AFPs. In the winter flounder, it has been suggested that the liver-type AFP genes evolved from their skin-type AFP counterparts (28). Thus, it is conceivable that the skin-type, or intracellular AFPs are an earlier evolutionary adaptation than the serum AFPs (liver-type), but liver-type AFPs are not necessarily derived from skin-type AFPs.
The production of lssAFP is up-regulated in the peripheral tissues such
as the gills and skin, which are likely the first tissues to encounter
environmental ice. It is now evident that skin-type AFPs are a
widespread biological phenomenon with their identification in three
species of fish. Moreover, with this work, it has been demonstrated
that the skin-type AFP is not restricted to the same biochemical
classification as the liver-type AFP found within the corresponding
fish species. However, a specific or direct biological function for an
intracellular AFP has yet to be determined, and the secretion of
skin-type AFPs outside the cell by nontraditional means has not been
ruled out. Nonetheless, the AFPs may have other functions aside from
ice crystal growth inhibition. For example, it has been demonstrated
that AF(G)Ps are capable of inhibiting membrane leakage during
phase-transition (29). Thus, one possibility is that the intracellular
AFPs work in conjunction with circulating AFPs to protect plasma
membranes at low temperatures and their membrane protective functions
may also be necessary to stabilize internal organelle membranes.
Clearly, identifying the physiological importance of the skin-type AFPs is a critical step in advancing the knowledge of marine fish
freezing protection.
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ACKNOWLEDGEMENTS |
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We thank Dr. Peter L. Davies for providing the antibody preparation, Mala Joshi for technical assistance, Rey Interior for amino acid analysis, and Shashikant Joshi for helpful discussions concerning tandem MS.
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FOOTNOTES |
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* This work was supported in part by the Medical Research Council (Canada) (to C. L. H.) and by the Natural Sciences and Engineering Research Council (Canada) (to G. L. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF306348.
¶ Recipient of an Ontario Graduate Scholarship.
§§ To whom correspondence should be addressed: Dept. of Biological Sciences, National University of Singapore, Singapore, 119 260. Tel.: 65-874-2699; Fax: 65-779-2486; E-mail: dbshead@nus.edu.sg.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M009293200
2 W-K. Low, Q. Lin, and C. L. Hew, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: AF(G)Ps, antifreeze (glyco)proteins (polypeptides); RT, reverse transcription; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; MS, mass spectrometry; UTR, untranslated region; ORF, open reading frame; HPLC, high performance liquid chromatography; bp, base pair(s)..
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