Type I antifreeze proteins expressed in snailfish skin are
identical to their plasma counterparts
Robert P. Evans and Garth L. Fletcher
Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada
Teleost fish that inhabit icy seawater synthesize anti-
freeze proteins ⁄ polypeptides (AFPs) or antifreeze glyco-
proteins (AFGPs) for protection against freezing.
Diverse species from numerous taxonomic groups pro-
duce AFPs that are grouped into four distinct classes
(types I, II, III and IV) based on their primary and
secondary structural characteristics [1–3]. Regardless of
protein structure, all fish AFPs lower the solution
freezing point noncolligatively by binding to certain
surfaces of ice crystals, modifying their structure and
inhibiting further growth. The difference between the
lowered freezing point and unaltered melting point is
termed thermal hysteresis and is used as a measure of
antifreeze activity [1,3,4].
Of the four classes of AFPs described thus far, the
simplest is type I AFP found in right-eye flounders
(Pleuronectes) and a few sculpin species (e.g. Myoxo-
cephalus). These polypeptides have high alanine con-
tent (> 60 mol%), have an amphipathic a-helical
secondary structure, and are usually quite small (3.3–
4.5 kDa) [2,5]. Until the past decade, it was generally
accepted that the synthesis of AFPs was confined
solely to liver tissue (termed liver type) for secretion
into blood for extracellular freeze protection. How-
ever, more recently, a novel subclass of type I
AFPs was isolated and characterized from the skin of
winter flounder Pseudopleuronectes americanus (for-

acid repeats or continuous hydrophobic face which typify the structure of
most other type I AFPs. These structural differences might have implica-
tions for their ice-crystal binding properties. These results are the first to
demonstrate a dual liver ⁄ skin role of identical type I AFP expression which
may represent an evolutionary intermediate prior to divergence into distinct
gene families.
Abbreviations
AFGPs, antifreeze glycoproteins; AFPs, antifreeze proteins ⁄ polypeptides; IBM, ice-binding motif; ORF, open reading frame;
UTR, untranslated region.
FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS 5327
are encoded by a separate subset of genes, were desig-
nated as skin-type AFPs. They are synthesized as
mature polypeptides that lack both signal and pro-
sequences, which suggests that they remain intracellu-
lar [6]. Recent publications of skin-type AFP isolation
from shorthorn (Myoxocephalus scorpius) and long-
horn (M. octodecemspinosus) sculpins indicate that the
production of AFP in peripheral epithelial tissues may
be a common trait in many fish species [7,8]. The char-
acterization of known skin-type AFPs and the presence
of antifreeze activity in skin tissues of other species has
led to the hypothesis that skin-type AFPs are wide-
spread ancestors of liver-type (plasma) AFPs [6,9].
Atlantic snailfish (Liparis atlanticus) and dusky
snailfish (L. gibbus) belong to a large family (Cyclo-
pteridae) of benthic and pelagic marine fishes that
inhabit northern regions of the Atlantic Ocean. Snail-
fish are closely related to sculpins, which belong to a
different family of the same order Scorpaeniformes
[10]. Both species spawn during the winter months in

screen using the open reading frame (ORF) of a
shorthorn sculpin skin cDNA as a probe. The
 260 bp clones (clone-c1 and clone-c2) contained
identical sequences, apart from a small difference in
the length of poly(A) tail and a few nucleotides at their
5¢ ends. However, the clones appeared to be truncated
versions of complete type I AFP messages. As indica-
ted by the underline in Fig. 1, one reading frame gave
an Ala-rich 26 amino acid peptide, that lacks an
obligatory in-frame start codon. This sequence infor-
mation was then used in 5¢-RACE reactions to ascer-
tain the remainder of the skin AFP cDNA sequence.
RNA ligase-mediated RACE was used to clone the
remaining 5¢ portion of the snailfish AFP cDNA. The
full L. atlanticus skin cDNA is 568 bp and contains a
complete 342 bp ORF (Fig. 1). The ORF encodes an
Ala-rich protein of 113 residues and was designated as
Las-AFP (L. atlanticus skin AFP). The putative start
and stop codons are underlined as well as three pos-
sible polyadenylation signal sequences [13].
The Las-AFP sequence was utilized to design appro-
priate RT-PCR and 3¢)5¢-RACE primers for de novo
cloning of AFP sequence from dusky snailfish skin
RNA. The 3¢-RACE procedure (primers indicated in
Fig. 2) produced a single band that was  450 bp,
whereas 5¢-RACE gave a 370 bp product. The overlap-
ping sequences were combined into a 587 bp clone
which contained a 342 bp ORF that encodes a 113
residue, Ala-rich, protein designated as Lgs-AFP
(L. gibbus skin AFP). The putative start and stop

skin tissue as well as a faint signal from gill is detect-
able with longer exposures. No other tissues gave
detectable signals on this northern blot. Similar results
were observed in another fish, except that there was a
Fig. 1. Nucleotide sequence and primary
translation product of L. atlanticus skin AFP
cDNA. The ORF is capitalized, whereas the
5¢- and 3¢-UTRs are in lower case letters.
The putative start and stop codons are
underlined in bold as are three possible
polyadenylation signal sequences. The
sequence obtained from the initial las-c1
and las-c2 cDNA clones are underlined.
RT-PCR or RACE primer sequences are
shown above (5¢fi3¢) or below (3¢fi5¢)
the nucleotide sequence. GenBank
Accession Number AY455862.
Fig. 2. Nucleotide sequence and primary
translation product of L. gibbus skin AFP
cDNA. The ORF is capitalized, whereas the
5¢- and 3¢-UTRs are in lower case letters.
The putative start and stop codons and the
polyadenylation signal are underlined.
RT-PCR or RACE primer sequences are
shown above (5¢fi3¢) or below (3¢fi5¢)
the nucleotide sequence. GenBank
Accession Number AY455863.
R. P. Evans and G. L. Fletcher Expression of snailfish type I AFPs in skin tissue
FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS 5329
definite detectable signal in liver tissue RNA (Fig. 3B).

Using a combination of cDNA library screening and
5¢-RACE, a complete cDNA corresponding to type I
AFP was cloned from Atlantic snailfish skin tissue and
Table 1. Amino acid composition (mol%) and molecular mass of snailfish type I AFPs.
Amino
acid
LaP-AFP
(protein)
LaS-AFP
(protein)
Las-AFP
(cDNA)
LgP-AFP1
(protein)
LgP-AFP2
(protein)
Lgs-AFP
(cDNA)
Asx 3.6 5.5 3 5.4 5.5 3
Glx 3.0 4.9 2 2.6 2.6 2
Ser 2.8 4.7 5 2.0 2.0 5
Gly 4.6 3.7 2 3.9 3.9 2
Arg 1.6 2.4 1 1.8 0.9 3
Thr 10.3 10.8 15 8.9 9.0 15
Ala 58.8 45.9 69 51.2 51.7 66
Pro 2.5 2.9 2 4.2 4.2 3
Val 5.6 4.9 5 8.4 8.5 5
Ile 1.3 2.1 1 1.7 1.8 1
Leu 2.6 4.1 2 2.3 2.3 2
Lys 3.4 4.1 5 6.6 6.6 5

Expression of snailfish type I AFPs in skin tissue R. P. Evans and G. L. Fletcher
5330 FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS
subsequently in closely related dusky snailfish. The
nucleotide and protein sequences are almost identical,
clearly suggesting that these AFPs shared a common
ancestral gene prior to snailfish species divergence.
This differs from taxonomically related shorthorn and
longhorn sculpin skin AFPs which produce quite con-
trasting proteins, whereas the UTRs of mRNA are
nearly identical [8].
Based on the cDNA sequence, both snailfish species
express 113 residue type I AFPs that are the largest
described to date. The predicted proteins lack signal or
pro-sequences, which indicates that the mature poly-
peptides remain intracellular. This would imply that
their location and function is analogous to the pre-
sumptive intracellular skin AFPs of winter flounder [6]
and sculpins [7,8]. However, the molecular mass of
snailfish skin proteins predicted from cDNA and their
N-terminal sequence are identical to the results deter-
mined for their purified plasma AFPs [11,12]. Further-
more, northern blots indicate that snailfish AFP
mRNA has consistently significant expression only in
skin tissue. Taken together, the evidence indicates that
the circulating plasma AFPs and skin localized AFPs
are identical proteins that are normally expressed by
the same skin-specific gene.
These results represent the first definitive report of
fish that synthesize identical AFPs for protection in
two different physiological locations. The assumption

intracellular antifreeze protection. Although the exact
subcellular location has not yet been unequivocally
established for skin-type AFPs, evidence from winter
flounder indicates that skin AFPs are present in gill
cell cytoplasm as well as in contact with the plasma
membrane outside epithelial cells [14].
Clearly, snailfish AFPs produced in epithelial cells
are secreted into blood to provide extracellular protec-
tion but it is not clear whether some protein remains
inside these cells. It is uncertain exactly how snailfish
AFPs are secreted from the cells that expresses them if
they do not contain the requisite signal sequences.
There have been recent reports of mature type I AFPs
being exported from cells in winter flounder epidermis
despite the absence of a secretion signal or pro-
sequence [14,15]. Furthermore, alternative pathways
for protein export that circumvent the usual endo-
plasmic reticulum–Golgi complex have been described
previously [16,17].
The northern blot experiments exhibited unexpected
variation in AFP expression patterns among individual
fish. Whereas skin tissues consistently produced high
levels of AFP mRNA, expression in liver ranged from
undetectable to high levels. This extreme individual
variation in mRNA expression has not been reported
previously for any species producing antifreeze. How-
ever, studies have shown geographic-dependent popu-
lation differences in antifreeze gene copy number
[18,19]. In fact, individual fish from one population of
Newfoundland ocean pout had demonstrable differ-

thermal hysteresis activity compared with other type I
AFPs [11]. Helical net and helical wheel representa-
tions (Fig. 6) indicate that Las-AFP contain none of
the ice-binding motifs (IBM) that were originally sug-
gested as important for ice binding [21–23]. Recently,
amino acid substitution experiments have determined
that it is the conserved Ala-rich hydrophobic surface
which is most important for ice-binding in type I AFPs
[5,24–26]. Las-AFP contain no full-length hydrophobic
surface is free from interfering polar residue side
chains. Furthermore, snailfish AFPs do not contain
Fig. 6. Schematic representations of Atlantic snailfish AFP secon-
dary structure. (A) Helical net, (B) helical wheel diagrams were
constructed by the
EMBOSS package located on the Canadian
Bioinformatics Resource web page. Hydrophilic residues DENQST
are marked with diamonds. Positively charged residues HKR are
marked with octagons. Aliphatic residues ILVM are marked with
squares.
Expression of snailfish type I AFPs in skin tissue R. P. Evans and G. L. Fletcher
5332 FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS
the requisite hydrogen bonding amino acids necessary
to create the elaborate terminal cap structures found in
most type I AFPs [21]. The lack of complete hydro-
phobic face and terminal caps might be responsible for
the low activity of these AFPs. It should be noted,
however, that the predicted structure of snailfish AFP
may not exactly correspond with structural data provi-
ded by experimental methods. It is possible that the
protein contains kinks or bends in the backbone

Tissue sample collection
Twelve Atlantic snailfish (L. atlanticus) were collected by
divers near Logy Bay, Newfoundland, in winter 2000. Two
specimens of dusky snailfish (L. gibbus) were collected from
Placentia Bay, Newfoundland during winter 1999. Tissues
were removed from anesthetized fish, immediately frozen in
liquid nitrogen and stored at )70 °C.
Skin library construction and screening
Total RNA from Atlantic snailfish skin tissue was isolated
using TRIzolÒ reagent (Invitrogen Canada Inc, Burlington,
ON, Canada) and poly(A)
+
mRNA was isolated from total
RNA using an Oligotex mRNA Kit (Qiagen Inc, Mississ-
auga, ON, Canada). A skin cDNA library was constructed,
as described by the manufacturer, using Lambda ZAPÒ II
library and ZAP-cDNAÒ Synthesis Kit and Gigapack
Ò
Gold III packaging extracts (Stratagene, La Jolla, CA,
USA). The primary skin cDNA library contained around
5 · 10
5
clones. Normally,  50 000 plaques were grown on
15 cm NZYCM plates for primary screening; 9 cm plates
were used in secondary and tertiary screens.
Hybond-N nylon membranes (Amersham Biosciences,
Piscataway, NJ, USA) were prepared and screened
Fig. 7. Classification of known type I AFP
sequences based on primary structural char-
acteristics. Amino acid sequence alignments

prior to use with ProbeQuant G-50 Micro Columns (Amer-
sham Biosciences). The final wash was performed in 1.0·
NaCl ⁄ P
i
, and 0.1% SDS, at 52 °C for 20 min. A 300 bp
DNA fragment corresponding to the ORF of shorthorn
sculpin skin (s3–2) clone [7] was used as a probe to screen
 2.0 · 10
5
clones of the primary cDNA library. Positive
plaques were first isolated and then pBluescriptÒ phage-
mids, to be used for sequencing inserts, were produced
using an in vitro excision protocol (Stratagene).
Northern blot analysis
Total RNA from various tissues of Atlantic and dusky
snailfish were isolated using TRIzolÒ reagent (Invitrogen
Canada Inc) as described by the manufacturer. Five-micro-
gram aliquots of total RNA were separated on 1% for-
maldehyde gels and analyzed by a nonradioactive northern
blotting procedure using positively charged nylon mem-
branes (Roche Diagnostics Canada, Laval, QC, Canada).
RNA was transferred to membranes using a VacuGene XL
Vacuum Blotting System (Amersham Biosciences) and
cross-linked with UV light. The membrane was hybridized
at 50 °C overnight in DIG Easy Hyb buffer (Roche Diag-
nostics). Probe was labeled with DIG-11–dUTP using a
DIG-High Prime DNA Labeling kit or in some cases with
a PCR DIG Probe Synthesis Kit (Roche Diagnostics) with
chemiluminescent signal detection using CDP-StarÒ. The
final wash was performed in 0.1· NaCl ⁄ P

Both 5¢- and 3¢-RACE reactions were performed using the
RNA ligase-mediated GeneRacer
TM
Kit, as described by
the manufacturer (Invitrogen Canada Inc). One microgram
of DNase-treated total RNA combined with Thermo-
script
TM
reverse transcriptase (Invitrogen Canada Inc) was
used to generate adapter-linked first strand cDNA for 1 h
in a 50 °C reaction. The first-strand cDNA was combined
with the appropriate primers and touchdown PCR amplifi-
cation was performed using DyNAzyme EXT
TM
DNA
polymerase (Finnzymes, Oy, Finland) in an Eppendorf
MastercyclerÒ. The touchdown cycling conditions consisted
of an initial 95 °C denaturing step (2 min), followed by 10
cycles of 94 °C (15 s), 72 °C decreased to 60 °C (15 s),
72 °C (60 s) and 25 more cycles of 94 °C (15 s), 60 °C
(15 s), and 72 °C (60 s). In order to obtain a product in
most reactions, dimethylsulfoxide was added at 10% (v ⁄ v).
RACE reaction products were separated on 1% agarose
gels and then purified using spin columns provided in the
kit GeneRacer
TM
Kit or by CONCERT
TM
Gel Extraction
System (Invitrogen Canada Inc). A TOPO TA CloningÒ

ics (Hospital for Sick Children, Toronto, ON, Canada).
Bioinformatics programs
Homologous nucleotide and protein sequences were
searched through blast searches on the NCBI web server.
The NCBI orf finder was utilized to identify putative
open reading frames in the nucleotide sequences. Helical
net and helical wheel diagrams were constructed using
emboss package located on the Canadian Bioinformatics
Resource web page (all located at .
gov/). Swiss PDB software used to generate a three-dimen-
sional model of Las-AFP. clustalx and treeview (1.6.1)
software were used to create an unrooted neighbor-joining
tree.
Acknowledgements
We thank M. King and Dr M. Shears at the OSC for
technical assistance and the OSC divers for sample col-
lection. We also thank Dr Ming Kao for help with
antifreeze activity measurements. This study was sup-
ported by a grant from NSERC.
References
1 Fletcher GL, Goddard SV, Davies PL, Gong Z, Ewart
KV & Hew CL (1998) New insights into fish antifreeze
proteins: physiological significance and molecular regu-
lation. In Cold Ocean Physiology (Po
¨
rtner HO & Playle
RC, eds), pp. 240–265. Cambridge University Press,
New York.
2 Ewart KV, Lin Q & Hew CL (1999) Structure, func-
tion and evolution of antifreeze proteins. Cell Mol Life

Publishing, Singapore.
10 Scott WB & Scott MG (1988) Atlantic Fishes of
Canada. University of Toronto Press, Toronto, ON.
11 Evans RP & Fletcher GL (2001) Isolation and charac-
terization of type I antifreeze proteins from Atlantic
snailfish (Liparis atlanticus) and dusky snailfish (Liparis
gibbus). Biochim Biophys Acta 1547, 235–244.
11a Evans RP & Fletcher GL (2005) Type I antifreeze pro-
teins: Possible origins from chorion and keratin genes
in Atlantic snailfish. J Mol Evol. doi: org/10.1007/
s00239-004-0067-y.
12 Evans RP & Fletcher GL (2004) Isolation and purifica-
tion of antifreeze proteins from skin tissues of snailfish,
cunner and sea raven. Biochim Biophys Acta 1700,
209–217.
13 Graber JH, Cantor CR, Mohr SC & Smith TF (1999)
In silico detection of control signals: mRNA-3¢-end-
processing sequences in diverse species. Proc Natl Acad
Sci USA 96, 14055–14060.
14 Murray HM, Hew CL, Kao KR & Fletcher GL (2002)
Localization of cells from the winter flounder gill
expressing a skin type antifreeze protein gene. Can J
Zool 80, 110–119.
15 Murray HM, Hew CL & Fletcher GL (2003) Spatial
expression patterns of skin-type antifreeze protein
in winter flounder (Pseudopleuronectes americanus)
epidermis following metamorphosis. J Morph 257,
78–86.
16 Mignatti P, Morimoto T & Rifkin DB (1992) Basic
fibroblast growth factor, a protein devoid of secretory

23 Zhang W & Laursen RA (1998) Structure–function
relationships in a type I antifreeze polypeptide. The
role of threonine methyl and hydroxyl groups in anti-
freeze activity. J Biol Chem 273, 34806–34812.
24 Baardsnes J, Kondejewski LH, Hodges RS, Chao H,
Kay C & Davies PL (1999) New ice-binding face for
type I antifreeze protein. FEBS Lett 463, 87–91.
25 Baardsnes J, Jelokhani-Niaraki M, Kondejewski LH,
Kuiper MJ, Kay CM, Hodges RS & Davies PL (2001)
Antifreeze protein from shorthorn sculpin: identification
of the ice-binding surface. Protein Sci 10, 2566–2576.
26 Fairley K, Westman BJ, Pham LH, Haymet ADJ,
Harding MM & Mackay JP (2002) Type I shorthorn
sculpin antifreeze protein. Recombinant synthesis,
solution conformation, and ice growth inhibition
studies. J Biol Chem 277, 24073–24080.
27 Marshall CB, Fletcher GL & Davies PL (2004) Hyper-
active antifreeze protein in a fish. Nature 429, 153.
28 Evans RP & Fletcher GL (2005) Type I antifreeze
proteins: possible origins from chorion and keratin
genes in atlantic snailfish. J Mol Evol in press.
Expression of snailfish type I AFPs in skin tissue R. P. Evans and G. L. Fletcher
5336 FEBS Journal 272 (2005) 5327–5336 ª 2005 FEBS