Many species of Antarctic and polar fish secrete macromolecular
antifreezes into their plasma in order to avoid freezing (DeVries,
1983; Davies and Hew, 1990). At present, four distinct types of
antifreeze proteins have been characterized from a variety of fish: the
antifreeze glycoproteins and three types of (AFPs) (
)(Davies
and Hew(1990) and Griffith and Ewart(1995) and references therein). The
antifreeze glycoproteins, which are found in three families of
Antarctic fish and polar cods, largely consist of a tripeptide repeat
(Ala-Ala-Thr) with a disaccharide attached to the threonyl residue.
Type I AFPs are alanine-rich,
-helical polypeptides found in many
righteye flounders and sculpins. Type II AFPs are enriched with
half-cystine and are found in sea raven, smelt, and herring. Type III
AFPs are globular proteins found in several Zoarcoid families including
eelpout and wolffish. Although the different AFPs and antifreeze
glycoproteins are structurally distinct, they share an unusual ability
to inhibit ice crystal growth by binding to the ice surface and thus
lowering the freezing temperature. At present, most if not all of the
antifreeze glycoproteins and AFPs have been isolated from serum, and
their DNA sequences have been deduced by cDNA cloning from the liver.
Invariably, all of these proteins described to date are synthesized as
larger precursor polypeptides containing the signal peptides, which is
consistent with a secretory role.
The AFP from the winter flounder, Pleuronectes americanus, has been studied extensively in terms
of its protein structure and function, gene organization, gene
expression, and regulation. The genome of the winter flounder contains
multiple copies of AFP genes, most of which are arranged as regular
tandem repeats (Scott et al., 1985).
The flounder AFP mRNAs
have recently been found in many tissues. In addition to liver, they
can be detected at relatively high concentrations in skin, scales, fin,
and gills. Furthermore, the liver and nonliver-derived AFP mRNAs
respond differently to seasonal and hormonal treatment (Gong et
al., 1992). Although the liver AFP mRNAs are tightly regulated
with a 1000-fold difference between their summer and winter levels, the
nonliver AFP mRNAs in skin, for example, exhibits only a modest
5-10-fold seasonal difference. Furthermore, in hypophysectomized
fish, the liver AFP mRNAs are induced more than 40-fold, with little
induction for the nonliver AFP mRNAs (Gong et al., 1995).
These experiments suggest that there may be distinct sets of AFP genes
that respond differently to a wide variety of stimuli. This prompted us
to re-examine the nature of the nonliver AFP mRNAs and their
presumptive gene products.
EXPERIMENTAL PROCEDURES
Materials
Winter flounder (P.
americanus) were collected from Conception Bay, Newfoundland,
Canada. Several tissues were removed and frozen in liquid nitrogen and
stored at -70 °C before use.
Isolation of Skin AFPs
Skin scrapings (46 g) were
homogenized in 500 ml of 0.1 M NH
HCO
using a Polytron homogenizer. After low speed centrifugation, the
supernatant was lyophilized (1 g), redissolved, and chromatographed in
a Sephadex G-75 column (2.5
80 cm) in 0.1 M NH
HCO
. Active fractions, measured with a
nanoliter osmometer, were pooled and rechromatographed once in the same
column. The repurified materials (approximately 50-100 mg) were
further fractionated on a Bondclone 10 C
column
(Phenomenex, Torrance, CA) using a 0.5% trifluoroacetic acid and
acetonitrile gradient. Individual fractions were pooled and
lyophilized.
Measurement of Antifreeze Activity
Antifreeze
activity was measured as thermal hysteresis (the difference between the
melting and freezing temperatures) essentially following the procedure
of Chakrabartty et al.(1989). Proteins were dissolved in 0.1 M NH
HCO
, and their concentrations were
determined by duplicate amino acid analyses. Activity measurements were
performed on a series of dilutions with concentrations ranging from 0.1
to 7.0 mM for each protein using a Clifton Nanolitre Osmometer
(Clifton Technical Physics, Hartford, NY). Prior to the series of
measurements for each protein, measurements were made from three sample
wells using buffer alone, and their average gave the background
hysteresis. For each dilution, measurements were made from three wells,
and the average was taken. The background hysteresis was then
subtracted from this value in order to obtain the antifreeze activity.
Structural Analysis of Skin AFP
Purified skin AFPs
from reverse phase HPLC were used for amino acid analysis, protein
sequencing, and mass spectroscopy. Both amino acid analysis and protein
sequencing were performed by the Biotechnology Service Centre, Hospital
for Sick Children, Toronto, and mass spectrometry was done by the
Carbohydrate Centre, University of Toronto, Toronto. For amino acid
analysis, the samples were hydrolyzed in 6 N HCl at 110 °C
for 24 h and analyzed using the Waters Picotag System. Because of the
blockage of the N-terminal methionine, skin AFPs were pretreated with
cyanogen bromide (in 5% formic acid, 24 h, with 200-fold molar excess
of CNBr) prior to protein sequencing in a Porton Gas Phase Sequencer.
Isolation of Skin AFP cDNA Clones
Total RNA was
isolated using the skin tissues containing scales and a dorsal fin from
a single individual fish collected in winter by the acid guanidium
thiocyanate-phenol-choloroform extraction method as originally
described by Chomczynski and Sacchi(1987) and modified for fish tissues
by Gong et al.(1992). Poly(A)
RNA was then
selected by oligo(dT)-Sepharose as described by Sambrook et
al.(1989). The cDNA library was constructed using the lambda
Uni-ZAP XR vector system from Stratagene (La Jolla, CA). About 2.9
10
primary clones were obtained from 2 µg of
poly(A)
RNA. The skin AFP cDNA clones were screened by
hybridization using a liver AFP cDNA clone, pkenc17, which encodes the
most abundant serum AFP component A or HPLC-6 (Pickett et al.,
1984). Because of the high representation of AFP clones in the skin
cDNA library, some AFP clones were obtained by colony hybridization
after bulk in vivo excision. For bulk in vivo excision, about 10
phages from amplified skin cDNA
library were used to infect plating bacteria together with the helper
phage ExAssist according to the manufacturer's manual
(Stratagene). About 200 colonies were transferred to a new plate and
hybridized with pkenc17 after transferring onto a nylon membrane,
Colony/Plaque Screen (Du Pont NEN). Both strong and weak hybridized
colonies were characterized for potential distinct AFP clones.
Hybridization was performed as described previously by Gong et
al.(1992) but washed at a less stringent condition (0.3 M NaCl, 60 °C).
DNA Sequencing
Double strand DNA sequencing was
performed by dideoxynucleotide chain termination using the T7 DNA
sequencing kit according to the manufacturer's instructions
(Pharmacia). Each clone contained about a 240-300-base pair cDNA
insert, and complete sequences were obtained by sequencing from both
ends using SK and T7 primers.
Primer Extension
A 22-mer oligonucleotide,
5`-GGCTGGTGCGTCCATGTTGATG-3`, complementary to the region starting 7
bases upstream of the ATG codon in sAFP2 (clone S3) (see Fig. 5), was synthesized by the Biotechnology Service Centre,
Toronto. The oligonucleotide was labeled using
[
-
P]ATP by T4 DNA kinase. The primer
extension experiment was carried out as described by Sambrook et
al.(1989).
Figure 5:
Sequence comparison of the pseudogene F2
and skin AFP cDNA clone S3. Identical nucleotides are shown as hyphens in S3. The two sequences encode an identical
polypeptide shown above the nucleotide sequences. The two major
transcription start sites, as determined by primer extension in Fig. 6, are indicated by carets. The sequence before
the transcription start sites are shown in lowercase letters.
The position of an intron is indicated by an arrowhead. The
putative TFIID binding site, aataat, the oligonucleotide for primer
extension, and the polyadenylation site AATAAA are underlined.
Figure 6:
Transcription start sites. Primer
extension was performed using a skin AFP-specific oligonucleotide as
described under ``Experimental Procedures.'' 5 µg of
scale total RNA and 30 µg of liver total RNA from the winter
flounder (WF) and 30 µg of total liver RNA from Atlantic
salmon (AS) were used as templates as indicated at each lane.
The size of primer extended products were determined by a set of
sequence ladders as shown on the left (nucleotides). The two
major extended products are indicated on the right.
Genomic Southern Blot Hybridization
Genomic DNA
was isolated from a testis collected from a single fish by the method
of Blin and Stafford(1976). Briefly, about 0.4 g of testis was digested
with proteinase K in 50 mM Tris, pH 8, 100 mM EDTA,
0.5% SDS overnight at 50 °C. The mixture was then extracted
extensively with phenol/chloroform. The aqueous phase was dialyzed
against TE (10 mM Tris, pH 8, 1 mM EDTA), and high
molecular weight DNA was collected by the addition of NaCl to 0.1 M and 2 volumes of ethanol. Restriction-digested genomic DNA was
separated by electrophoresis on a 0.7% agarose gel. The gel was soaked
in 0.4 M NaOH, 0.6 M NaCl for 30 min, and blotted
onto a Hybond membrane (Amersham Corp.) in the same solution.
Hybridization was performed as described previously (Gong et
al., 1992).
Northern Blot Hybridization
Total RNAs from
selected tissues were extracted as stated above. Total RNAs were
separated by formaldehyde agarose gel electrophoresis and Northern blot
hybridization was performed as described previously (Gong et
al., 1992). The relative levels of AFP mRNAs were estimated by
densitometric scanning of autoradiograms using ScanJet 3P (Hewlett
Packard) and analysis using a computer program (NIH Image 1.52).
RESULTS
Isolation of Skin Type AFP
The HPLC profile of
AFPs isolated from skin scrapings is shown in Fig. 1. The HPLC
profile of gel filtration chromatography-purified serum AFP is also
included for comparison. The serum AFPs contain only two major
components, HPLC-6 and HPLC-8 (Fig. 1A). Except for
HPLC-5, -7, and -9, which represent post-translational modifications
and minor serum AFPs, the other earlier elution peaks are not related
to AFPs, as examined by amino acid analysis (Fourney et al.,
1984). However, the HPLC profile of AFP isolated from the skin
scrapings was obviously more heterogenous and contained at least
5-6 components (Fig. 1B). The major skin AFPs are
designated as sAFP1, sAFP2, sAFP3, HPLC-6*, and HPLC-8*, respectively.
In addition, there were several peaks eluted early in the HPLC. These
early elution peaks were minimal in antifreeze activity. Furthermore,
amino acid compositions of these materials were not significantly
enriched with alanine and were not further investigated. However,
HPLC-6* and HPLC-8* had retention times identical to those of HPLC-6
and HPLC-8, respectively. Subsequent amino acid compositions and
protein sequencing analyses confirmed that they were in fact HPLC-6 and
HPLC-8, respectively, and indicated some contamination of the skin
samples from the blood.
Figure 1:
Reverse phase HPLC
profiles of flounder AFPs. A, serum AFP. B, skin AFP.
Sephadex G-75 purified antifreeze from sera (A) and skin
scrapings (B) were fractionated on a Bondclone 10 C18 column
(2.1
30 cm); the flow rate was 4.5 ml/min with a 20-40%
acetonitrile, 0.1% trifluoroacetic acid
gradient.
Amino acid analyses indicate that the skin
AFPs are enriched with alanine, with 60.2% of alanine comparable with
62.2% to HPLC-6 and HPLC-8 (data not shown). In addition, the skin AFPs
contain methionine and proline, two amino acid residues absent in serum
AFP, and are relatively low in leucine, aspartic acid, and glutamic
acids compared with HPLC-6 or HPLC-8. One of the skin AFPs (sAFP1)
contains histidine, which is absent in serum AFP. Unlike the serum AFP
(HPLC-6 and HPLC-8), the skin AFP N termini were blocked. sAFP1, sAFP2,
and sAFP3 were analyzed by atmospheric pressure ionization mass
spectroscopy to determine the nature of the blocking group. Based on
their cDNA sequences to be described in the later sections, these
peptides had M
of 3376.56, 3480.61, and 3482.67,
respectively. Atmospheric pressure ionization mass spectroscopy
analyses indicated that larger M
ions of 3419.06,
3480.61, and 3525.41 were present in sAFP1, sAFP2, and sAFP3,
accordingly. The difference (42 M
) was consistent
with the addition of an acetyl group at the N terminus. sAFP2, in
addition, contained a minor M
of 3366.87 and might
indicate slight contamination by sAFP7 (M
of
3324.46). Their amino acid sequences were confirmed by protein
sequencing after CNBr cleavage of the N-terminal methionine. Like the
serum AFPs, skin AFPs also contain similar threonine 11 amino acid
repeats. Interestingly, the skin AFPs resemble two previously sequenced
serum AFPs, SS-3 and GS-5 from the shorthorn sculpin and grubby
sculpin, respectively (Hew et al., 1985; Chakrabartty et
al., 1988), particularly in their N-terminal regions (Fig. 2). Both SS-3 and GS-3 are similarly blocked at the N
terminus by an acetyl group. In retrospect, the skin AFPs have
N-terminal structures more in line with the AFP isolated from the
shorthorn and grubby sculpins (Sicheri and Yang, 1995).
Figure 2:
Amino acid sequence comparison of
representative type 1 antifreeze polypeptides. HPLC-6, major
liver type AFP from winter flounder (Davies et al., 1982); sAFP1, skin type AFP from winter flounder (this report); SS3, AFP from shorthorn sculpin (Hew et al., 1985); GS5, AFP from grubby sculpin (Chakrabartty et al.,
1988); The empty spaces are to maximize sequence
identity.
In Fig. 3, activities of the skin AFPs plotted as a function of
concentration form hyperbolic curves consistent with those typically
observed for type I AFPs (Scott et al., 1987; Chakrabartty et al., 1989; Wen and Laursen, 1992a) and most other fish AFPs
(Kao et al., 1986). The activity curve of the major serum AFP,
HPLC-6, was in line with those previously obtained using a synthetic
analogue of this protein (Chakrabartty et al., 1989) and a
mixture of AFPs from flounder serum (Kao et al., 1986). In
contrast, the AFPs isolated from skin appeared far less active. The
skin AFPs displayed lower activities than the serum AFP in all but the
lowest (0.1 mM) concentration measured, and activity curves
appeared to be approaching saturation plateaus at lower concentrations
than the serum protein (Fig. 3). The curves for two of the skin
AFPs, sAFP2 and sAFP3, were virtually coincident with activities less
than half of that observed for HPLC-6. A detailed structural and
functional analysis of the skin type AFP will be reported elsewhere. (
)
Figure 3:
The concentration dependence of the
antifreeze activities of AFPs isolated from flounder serum and skin.
The AFPs are the major serum AFP (HPLC-6) and skin AFPs, sAFP2 and
sAFP3.
Isolation of Skin AFP cDNA Clones
A
winter flounder skin cDNA library was constructed using
Stratagene's lambda Uni-ZAP XR vector system. When a small
portion of the primary library was screened with a liver AFP cDNA clone
at low stringency (0.3 M Na
, 55 °C),
approximately 20% of the clones were positive, indicating an abundance
of the AFP mRNA in skin. 14 independent clones were completely
sequenced, and 9 distinct DNA sequences encoding 8 AFPs, sAFP1 to
sAFP8, were obtained (Fig. 4). The 14 cDNA clones are listed in
parentheses following each protein sequence. The cDNA clones encoding
the same sAFP have identical nucleotide sequence except for P12 and S3,
which have three single nucleotide substitutions. All these clones,
except for P13 and S6, which encode additional C-terminal sequences,
share a stronger sequence identity (91.7-99.2%) to each other
than to the liver AFP DNA sequences (72.1-82.2%). Because of the
obvious sequence difference of these skin clones and liver AFP cDNA
clones, we defined two sets of AFP genes, skin type and liver type AFP
genes, accordingly. Most of these skin AFP clones encode a short
polypeptide of 37-40 amino acid residues, and one of them (sAFP8)
has 54 residues instead (Fig. 4). The sAFPs are almost
identical, and the few amino acid differences are restricted to the
last few residues at the C-terminal end. However, unlike the liver type
AFP genes, which encode the secretory AFPs, the skin type AFP genes
encode mature polypeptides without the pre- and prosequences.
Figure 4:
Amino
acid sequence of flounder skin type AFP. The amino acid sequences were
deduced by cDNA and genomic clones (F2 and 11-3, from Davies and
Gauthier(1992)).
DNA
sequence comparisons indicate that the skin AFP clones are closely
related to two previously identified AFP genomic sequences, F2 and
11-3 (Davies and Gauthier, 1992). Part of the F2 sequence is
shown in Fig. 5. The sequence of 11-3, which is almost
identical to that of F2, in the region shown in Fig. 5had only
5 nucleotide changes and a 5-base deletion (Davies and Gauthier, 1992).
Due to the presence of in frame stop codons in the 5` upstream region
corresponding to the presequence in the liver AFP gene and the lack of
a typical TATA box in the putative promoter region, these two genes
have been previously assigned as pseudogenes (Davies and Gauthier,
1992). As shown in Fig. 4, sAFP2 is identical to the protein
encoded by the ``pseudogene'' F2 and is different from the
AFP encoded by 11-3 by one amino acid. One of the cDNA clones
encoding sAFP2 (S3) has only two nucleotide substitutions compared with
F2 (Fig. 5). These substitutions occur only in the
3`-untranslated region. Another cDNA clone encoding sAFP2 (P12) also
has two nucleotide substitutions at different locations (data not
shown). Alignment of the cDNA and the genomic sequences indicate that
there is an intron between nucleotides 167 and 168. Therefore, it is
likely that both F2 and 11-3 code for functional genes in skin
and other peripheral tissues.
In order to confirm the isolated
clones contain full-length coding sequences and to map the
transcription start site of the skin type AFP genes, primer extension
was carried out as shown in Fig. 6. There are two major
extension products using skin total RNA as templates. The extension is
17 or 18 base pairs from the 5` end of S3 cDNA clones. Only one
extension product corresponding to the long extension product in the
scales was detected using the liver RNA as templates. The two extension
products map the transcription start site at bases 130 and 131 of the
F2 gene (Fig. 5). A putative TFIID binding motif AATAAAT is
found 25 nucleotides upstream of the first start site, further
indicating that F2 and 11-3 are functional. These data also
confirm that the skin AFP clones contain a full-length coding sequence,
without the signal peptide and prosequences. The two extension products
may indicate two transcriptional initiation sites for the skin type AFP
genes. Alternatively, because the primers we used hybridize to all skin
AFP genes, the two extension products could be the result of genetic
polymorphism.
Multiple Copies of Skin Type AFP Genes in the
Genome
Because 9 different skin AFP cDNA clones encoding eight
distinct skin type AFPs were identified, it is likely that the skin
type AFP genes belong to a multigene family similar to the liver type
AFP genes. In order to confirm this, a genomic blot hybridization was
performed. Genomic DNA was isolated from the testis collected from a
single individual fish and cut with four different restriction enzymes, HindIII, EcoRI, SstI, and BamHI.
Two identical blots were made and probed with the liver type (pkenc 17)
and skin type (S3) AFP probes, respectively. As shown in Fig. 7,
both probes hybridize to multiple bands. It is interesting to note that
the skin probe recognizes most, if not all, liver AFP fragments,
whereas the liver AFP probe only hybridized to a limited number of DNA
fragments. Because most of the liver AFP genes are regular tandem
repeats and most liver AFP fragments contain multiple copies of AFP
genes (up to 40 copies), it is likely that even a weak hybridization
from the skin probe produces a strong signal. The cross-hybridization
was also observed in the Northern blot analyses (Fig. 8A). Another possibility is that some skin type
AFP genes may be clustered together with the liver type AFP genes. The
skin AFP-specific fragments can be recognized by the comparison of the
two blots as shown in Fig. 7and are indicated by dots. Most of
the skin type AFP genes are also most likely linked as indicated by the
hybridization pattern in BamHI-digested DNA, in which the skin
type AFP gene signals are restricted to two high molecular weight
fragments (over 23 kilobases). These data, together with the
identification of at least nine distinct skin AFP cDNA sequences,
unequivocally support the presence of a skin type AFP multigene family.
The hybridization pattern of the liver probe is similar to the
previously published genomic blot by Scott et al.(1985), which
estimated there were about 40 copies of AFP genes in the winter
flounder genome. This estimation probably did not include the skin type
AFP genes. By comparison of the hybridization intensity of skin and
liver AFP DNA fragments, the number of skin type AFP genes is almost
the same as the number of liver type AFP genes. Therefore, it is likely
that the skin type AFP genes constitute an additional 30-40
copies of AFP genes in the genome. So far, we have isolated nine
different skin AFP cDNA clones encoding eight distinct AFPs. At the
protein level, at least three different antifreeze polypeptides have
been identified by reverse phase HPLC.
Figure 7:
Genomic Southern blot analysis of AFP gene
family. The genomic DNA was prepared from a single individual and cut
by four different restriction enzymes: HindIII, EcoRI, SstI, and BamHI. Two identical blots
were made and probed with the inserts of skin clone S3 and the liver
clone pken17, respectively. The skin-specific fragments as indicated by carets are defined as those only present in the skin blot or
weakly in the liver blot. Molecular weight marker lambda
DNA/HindIII (Life Technologies Inc.) are shown on both the left and the right sides.
Figure 8:
Tissue distribution of AFP mRNAs by
Northern blot analysis. Total RNAs were prepared from eight different
tissues as indicated, separated by electrophoresis, blotted, and probed
with the skin (S3, A) and liver (pkenc, B) AFP probes
respectively. 10 µg of total RNA was loaded on each lane except for
the liver RNA in B, which was loaded with 1 µg. C, the blot was probed with a mixture of liver and skin AFP
probes. The positions of liver type and skin type AFP mRNAs are
indicated.
Tissue Distribution of AFP Transcripts
The tissue
distribution of AFP transcripts were previously examined using a liver
cDNA probe in the winter flounder (Gong et al., 1992). Because
the hybridization was performed at a relatively low strigency, the
nonliver AFP transcripts were likely to be detected due to
cross-hybridization. With the availability of the skin AFP probe, the
tissue distribution of AFP mRNAs were re-examined using a high
stringency wash condition (0.015 M NaCl, 72 °C).
Preliminary experiments in which the liver and skin cDNA clones were
cross-hybridized indicated that this wash condition resulted in a
minimal cross-hybridization between the skin and liver genes. As shown
in Fig. 8, total RNAs were isolated from selected tissues
collected from a single fish and probed with the skin and liver AFP
probes, respectively. The skin type AFP mRNAs were detected in all
tissues examined, including the liver, stomach, intestine, heart,
spleen, and kidney but were strongly expressed in the exterior tissues
such as skin, scales, fin, and gills (Fig. 8A). These
last four tissues expressed similar levels of skin type AFP mRNAs,
whereas the other tissues expressed 1-10% of the level found in
these exterior tissues. Because the AFP cDNA clones represent 20% of
the clones in the skin cDNA library, AFP mRNAs are likely an abundant
mRNA species in all tissues in the winter. In the exterior tissues, the
level of AFP mRNAs are extraordinally high. It is also interesting that
the two types of AFP mRNAs can be distinguished by their size, because
the liver type AFP mRNAs are slightly larger than the skin type because
the liver type AFP gene contains approximately 132 additional
nucleotides encoding for the pre- and prosequences. In the liver lane (Fig. 8A), the upper signal is probably due to a
cross-hybridization of the skin probe to the extremely abundant liver
type AFP mRNAs, which constitute about 0.5-1% of total RNA
(Pickett et al., 1983). Nevertheless, the skin type AFP mRNAs
can be clearly detected as the lower band. As shown in Fig. 8B, the expression of liver type AFP mRNAs is
restricted to liver and intestine and possibly the stomach. To compare
the total levels of AFP mRNAs in the liver and skin tissues, a blot
containing liver, scale, and stomach total RNAs was hybridized with a
mixture of liver and skin AFP probes that were mixed and labeled in the
same reaction to ensure the same specific activity for the two probes (Fig. 8C). The level of scale AFP mRNA is about
10-20% of the liver level, indicating that the skin and other
exterior tissues such as gills are also major sites of AFP synthesis.
In comparison, the stomach mainly expressed the skin type AFP mRNAs at
a level of about 10% of that in scales (Fig. 8C).To
further demonstrate the presence of skin type AFP mRNA in the liver, a
more specific approach, primer extension, was employed using a skin
AFP-specific oligonucleotide. As shown in Fig. 6, a clear
extension signal from the liver RNA, which is identical to one of the
two major extension signals from the scale RNA, was detected,
indicating that the skin type AFP mRNAs are indeed present in the
liver. The relative level of skin type AFP mRNA in the liver, based on
the intensity of the extension signals and inputs of RNA was estimated
to be at least 100-fold lower than that observed in the scales, which
has the highest expression level for the skin type AFP genes. Liver RNA
from Atlantic salmon, a species without the AFP genes, was used as a
control. It has no detectable extension product, indicating the high
specificity of the primer used.
DISCUSSION
In the present study, we have isolated a family of skin type
AFPs and their corresponding cDNA genes. The skin type AFP genes are
ubiquitously expressed in all tissues examined, with high levels in the
exterior tissues such as skin, scales, fin, and gills. Structurally,
the skin type AFP lacks both the pre- and prosequences and might imply
an intracellular role in freeze protection. These studies confirm and
extend our previous suggestion that there are at least two different
sets of AFP genes, i.e. liver type and skin type, in the
winter flounder (Gong et al., 1992, 1995).
Function of Skin Type AFPs
In almost all AFP-producing
fish species studied, AFPs are synthesized in the liver and secreted
into the blood circulation. The AFPs thus function through lowering the
plasma freezing temperature to prevent fish from freezing in ice-laden
sea water. The presence of the skin type AFPs provides an additional
twist to our understanding of the function of AFPs. Because the skin
AFP genes encode polypeptides without the signal peptide, they may
function as intracellular proteins, although the possibility that these
small polypeptides use an alternate pathway for secretion cannot be
presently ruled out (Mignatti et al., 1992). However, analysis
of the HPLC profile (Fig. 1A) of the plasma AFP did not
reveal the presence of any skin type AFP in the circulation and thus
would argue against any significant secretion of the skin type AFPs.
The intracellular localization of AFPs may imply an unusual role in the
protection of fish from freezing. A cytosolic AFP could play a
significant antifreeze role by providing a barrier to ice crystal
passage or growth through the skin. Valerio et al.(1992) found
that isolated skin from winter flounder was a significant barrier to
ice crystal growth, which would highlight a significant protective
function of the skin type AFPs. The very high levels of skin type AFP
message in exterior tissues (i.e. 20% of total skin mRNA) may
reflect a need for high concentrations of AFP to achieve effective
protection from freezing. In many interior tissues, the expression is
relatively low but significant, about 1-10% of the skin level.
These tissues may already be protected by circulating plasma AFPs that
are produced in liver. Low levels of intracellular AFPs may also have
other physiological roles in these tissues.
Activity of the Skin AFP
The activity of the fish
skin AFPs falls within the range of fish AFP activities in general (Kao et al., 1986). However, they are not as active as the serum
AFP from winter flounder (Fig. 3). The presence of AFPs with
different activities within a species is not without precedent.
Comparison of AFPs from the shorthorn sculpin revealed that the longer
45-residue AFP, SS-8, is highly active, with an activity close to that
of winter flounder serum AFP, whereas the curve for the shorter
32-residue AFP, SS-3, from the same species is much lower (Kao et
al., 1986) and consistent with those of the winter flounder skin
AFPs, sAFP2 and sAFP3.The skin AFPs lack several residues that, in
the serum AFP, are known to contribute to activity (Wen and Laursen,
1992b). Most of the skin AFPs have only Thr residues as potential ice
crystal interaction sites because their helical repeats are generally
Thr-Xaa
instead of
Thr-Xaa
-(Asn/Asp)-Xaa
. The identification of
specific ice-binding motifs in the crystal structure of flounder serum
HPLC-6 consisting of Thr/Asp or of Thr/Asn/Leu (Sicheri and Yang, 1995)
further defines the functional implications of a Thr-only AFP sequence.
Single Thr residues are identified as incomplete motifs that would
presumably bind more weakly to ice. The proposed structural basis for
differences among the activities of the AFPs isolated from skin and
serum is also consistent with findings for other type I AFPs. An AFP
containing four sequence repeats was isolated from yellowtail flounder (P. ferrugineus), but its activity, on a molar basis, was
equivalent to the shorter 3-repeat flounder serum AFP (Scott et
al., 1987). The failure of an extra repeat to increase activity
was attributed to the presence of single Thr residues (incomplete
ice-binding motifs) in three of the four repeats.
Evolution of Type I AFP Genes
It is clear that
both liver and skin type AFP genes belong to multigene families. Most,
if not all, of the liver genes appear to be tandemly repeated with
regular spacing (Scott et al., 1985). The skin type AFP genes
(30-40 copies) appear to be also linked. The ubiquitously
expressed skin type AFP genes may represent a primitive AFP gene in
this multi-gene family. Gene duplication of the primordial skin type
AFP genes and subsequent divergence or selection may give rise to two
distinct AFP genes, i.e. liver type and skin type. One skin
type AFP gene may have gained a signal peptide sequence, prosequence,
and liver regulatory elements to become a liver-specific gene encoding
secreted AFPs. Selection during ocean cooling events may then have led
to the amplification of genes encoding highly active AFP forms
expressed in liver. The emergence of a liver-specific AFP gene and its
subsequent amplification were likely relatively recent events and
occurred after the divergence of the winter flounder and sculpins,
which also produce type I alanine-rich AFPs.Although the type II
fish AFPs found in three different taxonomic groups are homologous to
the C-type lectins and appear to have evolved from the
carbohydrate-recognition domains of these proteins (Davies et
al., 1993; Ewart and Fletcher, 1993), the evolutionary origins of
the other fish AFPs remain unclear. The type I AFPs do not appear to be
homologous to known protein families. However, many proteins with the
structural characteristics of the skin type AFPs are present in the
lower vertebrates and the invertebrates. For example, the skin from
many amphibians contains antimicrobial proteins that form small
amphiphilic helices (Kreil, 1994). If similar proteins occur in fish
skin, they may be progenitors of the type I AFPs or they may be among
the proteins that we now identify as skin type AFPs. These
possibilities and their implications for the roles and properties of
the type I AFPs should be examined.
Differential Expression of AFP Gene Family
There
are about 70-80 copies of AFP genes including both the liver and
skin type in the winter flounder genome. The liver type AFP genes
appear to be expressed predominantly in liver and to a much lesser
extent in intestine. Consistent with this, we have not isolated any
liver type AFP cDNA clones from the skin cDNA library. In contrast, the
skin AFP genes are ubiquitously expressed. Because there are
30-40 copies of skin AFP genes, whether or not some of these skin
type genes are specifically expressed in certain tissues remain a
question. It will be interesting to characterize AFP cDNA clones from
other tissues such as gills, heart, kidney, and spleen. The discovery
of nonliver AFP genes in the winter flounder suggests that variants
with different structures and tissue specificities may occur in the AFP
gene families of other AFP-producing species, particularly those
species producing different types of AFPs. For example, like the winter
flounder, the ocean pout (Macrozoarces americanus), which
synthesizes type III AFPs, has an AFP multigene family (Hew et
al., 1988) and expresses AFP mRNAs predominantly in the liver but
also in many nonliver tissues (Gong et al., 1992). It will be
interesting to determine whether there are different sets of AFP genes
with modified or different structures and functions in this species. Our observation, to our knowledge, represents the first report that
two distinct types of AFPs are present within the same species and has
raised new questions regarding the evolution and gene regulation of
these proteins. Our success in identifying the new skin type AFP will
stimulate further research on the identification of similar AFPs in
other fish species and the role of these AFPs in intracellular
function.