Isolation and Characterization of Skin-type, Type I Antifreeze Polypeptides from the Longhorn Sculpin, Myoxocephalus octodecemspinosus*

Woon-Kai LowDagger §, Qingsong LinDagger ||, Costas StathakisDagger , Ming MiaoDagger §, Garth L. Fletcher**, and Choy L. HewDagger §||Dagger Dagger §§

From the Dagger  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 Dagger Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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).

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tissue Sample Collection-- Longhorn sculpin was collected from Conception Bay, Newfoundland, Canada. Tissues were collected on Mar. 6, 1996, and stored at -70 °C before use.

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 [alpha -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.

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+/- (MBI Fermentas Inc., Flamborough, ON).

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 [alpha -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.

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-beta -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<UP><SUB>4</SUB><SUP>+</SUP></UP>)SO<UP><SUB>4</SUB><SUP>−</SUP></UP> and pelleted. The pellet was re-suspended in 0.1 M ammonium bicarbonate and desalted on a SephadexTM G-25 (Amersham Pharmacia Biotech) (2.5 cm × 60 cm) column with 10 mM Tris-HCl (pH 7.5). Protein fractions were loaded onto a SP-Sephadex C-25 (Amersham Pharmacia Biotech) (1.5 cm × 10.5 cm) column and eluted with 1 M NaCl in 10 mM Tris-HCl (pH 7.5). The bound fraction was collected and final purification was achieved by reverse-phase HPLC using a Jupiter 10-µm C4 300 Å (250 mm × 21.20 mm) column (Phenomenex, Torrance, CA).

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



View larger version (52K):
[in this window]
[in a new window]
 
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).

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 alpha -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.



View larger version (39K):
[in this window]
[in a new window]
 
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' right-arrow 3'), or below (3' right-arrow 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.

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).



View larger version (40K):
[in this window]
[in a new window]
 
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.

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).



View larger version (19K):
[in this window]
[in a new window]
 
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.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Amino acid analysis results for r-lssAFP(38) and lssAFP-7 to -10 
Key type I AFP residues are highlighted in grey. The percent Ala composition, and the molecular mass of the major monoisotopic peak of each species as determined by nanospray ESI MS are given below. Values indicated by an asterisk (*) are predicted values from the cDNA sequence for lssAFP.

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.



View larger version (25K):
[in this window]
[in a new window]
 
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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>+</SUP></UP> through acetylation may explain why the native lssAFPs did not bind to the strong cation exchange resin as expected. The identification of a type I AFP within the longhorn sculpin, which produces the type IV serum AFP (5-7), represents the first instance, to our knowledge, where one species of fish produces two different biochemical classes of antifreeze proteins.



View larger version (54K):
[in this window]
[in a new window]
 
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.



View larger version (106K):
[in this window]
[in a new window]
 
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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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.


    ABBREVIATIONS

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)..


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Davies, P. L., and Hew, C. L. (1990) FASEB J. 4, 2460-2468[Abstract/Free Full Text]
2. Davies, P. L., and Sykes, B. D. (1997) Curr. Opin. Struct. Biol. 7, 828-834[CrossRef][Medline] [Order article via Infotrieve]
3. DeVries, A. L. (1983) Annu. Rev. Physiol. 45, 245-260[CrossRef][Medline] [Order article via Infotrieve]
4. Feeney, R. E., Burcham, T. S., and Yeh, Y. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 59-78[Medline] [Order article via Infotrieve]
5. Deng, G., Andrews, D. W., and Laursen, R. A. (1997) FEBS Lett. 402, 17-20[CrossRef][Medline] [Order article via Infotrieve]
6. Deng, G., and Laursen, R. A. (1998) Biochim. Biophys. Acta 1388, 305-314[Medline] [Order article via Infotrieve]
7. Zhao, Z., Deng, G., Lui, Q., and Laursen, R. A. (1998) Biochim. Biophys. Acta 1382, 177-180[Medline] [Order article via Infotrieve]
8. Yeh, Y., and Feeney, R. E. (1996) Chem. Rev. 96, 601-618[CrossRef][Medline] [Order article via Infotrieve]
9. Hew, C. L., and Yang, D. S. (1992) Eur. J. Biochem. 203, 33-42[Abstract]
10. Gong, Z., Ewart, K. V., Hu, Z., Fletcher, G. L., and Hew, C. L. (1996) J. Biol. Chem. 271, 4106-4112[Abstract/Free Full Text]
11. Low, W. K., Miao, M., Ewart, K. V., Yang, D. S., Fletcher, G. L., and Hew, C. L. (1998) J. Biol. Chem. 273, 23098-23103[Abstract/Free Full Text]
12. Cheng, C. H. (1998) Curr. Opin. Genet. Dev. 8, 715-720[CrossRef][Medline] [Order article via Infotrieve]
13. Lin, Q., Ewart, K. V., Yan, Q., Wong, W. K., Yang, D. S., and Hew, C. L. (1999) Eur. J. Biochem. 264, 49-54[Abstract/Free Full Text]
14. Lin, Q., Ewart, K. V., Yang, D. S., and Hew, C. L. (1999) FEBS Lett. 453, 331-334[CrossRef][Medline] [Order article via Infotrieve]
15. Chakrabartty, A., Yang, D. S., and Hew, C. L. (1989) J. Biol. Chem. 264, 11313-11316[Abstract/Free Full Text]
16. Hew, C. L., Fletcher, G. L., and Ananthanarayanan, V. S. (1980) Can. J. Biochem. 58, 377-383[Medline] [Order article via Infotrieve]
17. Hew, C. L., Joshi, S., Wang, N. C., Kao, M. H., and Ananthanarayanan, V. S. (1985) Eur. J. Biochem. 151, 167-172[Abstract]
18. Chakrabartty, A., Hew, C. L., Shears, M., and Fletcher, G. (1988) Can. J. Zool. 66, 403-408
19. Sicheri, F., and Yang, D. S. (1995) Nature 375, 427-431[CrossRef][Medline] [Order article via Infotrieve]
20. Yang, D. S., Sax, M., Chakrabartty, A., and Hew, C. L. (1988) Nature 333, 232-237[CrossRef][Medline] [Order article via Infotrieve]
21. Harding, M. M., Ward, L. G., and Haymet, A. D. (1999) Eur. J. Biochem. 264, 653-665[Abstract/Free Full Text]
22. Wen, D., and Laursen, R. A. (1992) J. Biol. Chem. 267, 14102-14108[Abstract/Free Full Text]
23. Cheng, A., and Merz, K., Jr. (1997) Biophys. J. 73, 2851-2873[Abstract]
24. Baardsnes, J., Kondejewski, L. H., Hodges, R. S., Chao, H., Kay, C., and Davies, P. L. (1999) FEBS Lett. 463, 87-91[CrossRef][Medline] [Order article via Infotrieve]
25. Chakrabartty, A., and Hew, C. L. (1991) Eur. J. Biochem. 202, 1057-1063[Abstract]
26. Lipman, D. J. (1997) Nucleic Acids Res. 25, 3580-3583[Abstract/Free Full Text]
27. Duret, L., Dorkeld, F., and Gautier, C. (1993) Nucleic Acids Res. 21, 2315-2322[Abstract]
28. Davies, P. L., and Gauthier, S. Y. (1992) Gene (Amst.) 112, 171-178[Medline] [Order article via Infotrieve]
29. Hays, L. M., Feeney, R. E., Crowe, L. M., Crowe, J. H., and Oliver, A. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6835-6840[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.