Cloning, characterization and expression analysis of interleukin-10
from the common carp,
Cyprinus carpio
L.
Ram Savan
1
, Daisuke Igawa
2
and Masahiro Sakai
2
1
United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto, Japan;
2
Faculty of Agriculture,
Miyazaki University, Miyazaki, Japan
Interleukin (IL)-10 was cloned from the common carp
(Cyprinus carpio L.) using IL-10 primers from carp head
kidney following stimulation with concanavalin A and
lipopolysaccharide. The cDNA consisted of a 1096 bp se-
quence containing a 55 bp 5¢ untranslated region and a
498 bp 3¢ untranslated region. An open reading frame of
543 bp encoded a putative 180 amino acid protein with a
putative signal peptide of 22 amino acids. The signature
motif of IL-10 is conserved in carp sequence. A 2083 bp
genomic sequence of carp IL-10 was found to contain five
exons interrupted by four introns. With the exception of
much more compact introns, the genomic structure was
similar to that of mammalian IL-10. By homology, phylo-
geny and genomic analyses, the carp gene cloned was des-
ignated as IL-10. Carp IL-10 was expressed in head, kidney,
liver, spleen and intestine during the resting phase. The gene

factor-a [12], transforming growth factor [13,14], IL-8 [15]
andIFN[16],haveallbeenclonedinfish.
Recently, IL-10 homologues from torafugu (Taki-
fugu rubripes) and spotted green puffer fish (Tetraodon
nigroviridis) have been submitted to the EMBL database
(accession numbers CAD62446 and CAD67773), facilita-
ted by the fugu sequencing project [17]. However, expres-
sion of IL-10 has not been reported in fish. This is the first
report of an investigation of the expression patterns of IL-
10 in fish, in different tissues and its inducibility, when
stimulated with lipopolysaccharide (LPS). The presence of
IL-10 in fish gives significant insight on the regulation of
the immune response in fish. By homology, phylogeny and
genome analyses, the carp gene cloned was confirmed as
IL-10.
Materials and methods
Fish
Common carp (mean weight 100 g) was obtained from
Sunaso fisheries farm (Miyazaki, Japan). The fish were
acclimatized in an aerated fresh water tank at 20 °C, under
a natural photoperiod, and fed for 2 weeks, prior to use in
the study.
Cloning and characterization of the carp IL-10 gene
A carp cDNA library, produced following stimulation with
concanavalin A and LPS [18], was used to isolate the IL-10
gene, employing IL-10-Fw2 and IL-10-Rv2 primers
(Table 1), which were designed based on the conserved
regions of puffer fish and mammalian IL-10. PCR was
performed using a PTC-200 (MJ Research, Waltham, MA,
USA) with 30 reaction cycles of: 30 s at 94 °C, 30 s at 58 °C

; Takara Bio Inc,
Japan), 5 lL of each gene-specific primer and 27.5 lL
of water. The products obtained were cloned into the
PGEM-T Easy vector (Promega, USA) and transformed
into JM109 by electroporation (BTX 399; Genetronics, San
Diego, CA, USA).
Sequence and phylogenetic analyses
The sequences were compared with those in the database by
using the
BLASTX
algorithm [20]. The signal sequences were
predicted using the
SIGNAL IP
program. Protein alignment
and percentage identities were calculated by
CLUSTAL W
using
BIOEDIT
software [21]. Hydropathy analyses of carp,
torafugu and human IL-10 amino acid sequences were
carried out [22]. Phylogenetic analysis was carried out for
the deduced amino acid sequences of carp and other IL-10
homologues. Phylogenetic trees were obtained by the
neighbor joining method, with 1000 replications to obtain
Bootstrap values, using
PAUP
software [23]. IFN-c was used
as an outgroup in this analysis.
Expression of the IL-10 gene
Analysis of IL-10 gene expression in healthy tissues by

1640 supplemented with 10% carp serum and 1% strepto-
mycin/penicillin. Controls of the same cells were incubated
for 0, 1, 3 and 6 h in identical medium but without LPS.
Total RNA extracted from cells after culture was used for
cDNA synthesis (ReverTra Dash; Toyobo).Semiquantita-
tive analysis was carried out according to the method
described by Laing et al. [24]. In order to adopt a more
semiquantitative approach for analysing IL-10 gene expres-
sion, both carp IL-10 and b-actin genes were amplified using
a range (21–30) of PCR cycles. Following this procedure, an
optimal number of PCR cycles (24 for IL-10 and 21 for b-
actin) was determined and subsequently employed in the
above expression analysis. The carp IL-10 gene/b-actin ratio
was determined by densitometry, performed by measuring
the photo-stimulated luminescence values using
SCIENCE
LAB
99
IMAGE GAUGE
software (Fujifilm, Tokyo, Japan)
and by comparing the carp IL-10 transcript levels with those
of b-actin.
PCR conditions
All PCR reactions were performed according to the
following protocol: 1 lL of cDNA was mixed with 5 lL
of dNTPs (10 l
M
of each dNTP), 10 · Gene Taq Universal
buffer, 0.5 lLofTaq polymerase (5 UÆmL
)1

molecular mass polypeptide with a pI of 7.89. A similar
Fig. 2. Alignment of the deduced amino acid sequence of carp IL-10 with other homologues in pufferfish and mammalian counterparts. Identical amino
acid residues are indicated by dashes (–), while dots indicate gaps that have been introduced for optimal alignment. IL-10 signature motifs are
shown in the box. The position of A–F helices that has been determined in human IL-10 is shown as a ladder in the alignment. Cysteines from the
matured proteins forming disulphide bonds are denoted as numbers relative to their positions. The arrowheads depict the residues important for
the structural core of the IL-10 gene. The underlined amino acid residues are the signal sequences of the respective genes. The asterisk indicates the
residues important for an interaction with the IL-10R/R1 receptor chain. The accession numbers of the IL-10 sequences used in the alignment are as
follows: human, NP_000563; cat, AAC64708; rat, CAA43090; mouse, A34853.
Ó FEBS 2003 Cloning and analysis of IL-10 in fish (Eur. J. Biochem. 270) 4649
hydropathy profile was observed in torafugu and carp IL-
10 sequences (Fig. 3). The sequence is deposited with
DDBJ under the accession number AB110780.
Genomic structure of carp IL-10
Using the carp primers IL-10-Fw2 and IL-10-Rv2, we
amplified a product of 1.4 kbp from carp genomic DNA.
A 1403-bp IL-10 genomic sequence was obtained upon
sequencing of the cloned product using T7 or SP6 primers
(Fig. 1). The carp IL-10 is composed of five exons and four
introns, and thus similar to its mammalian counterparts.
The exons were positioned exactly to the exons in
mammalian IL-10. Typical intron splice motifs were
observed at the 5¢ (GT) and 3¢ (AG) ends of each intron.
The four introns are 150, 242, 444 and 119 bp in length,
respectively. However, while the mammalian counterpart
spanned > 5.5 kb, the carp IL-10 was only  2 kb.
Structural and phylogenetic analyses of carp IL-10
Alignment of the deduced amino acid sequence of IL-10
with those of mammalian homologues revealed common
structural features (Fig. 2). The IL-10 signature sequence
motif [KQS]-x(4)-C-[QYC]-x(4)-[LIVM](2)-x-[FL]-[LMV]-

head kidney and liver tissues was carried out using a time
course in vitro stimulation with LPS (Fig. 6). A relative
increase of IL-10 gene expression was seen at 1 h poststim-
ulation and showed a slight decrease at 3 h, producing a low
intensity product at 6 h in head kidney. In liver, the IL-10
Fig. 3. Hydropathy plot of putative IL-10 proteins from carp, torafugu
and human. The x-axis denotes the residue position and the y-axis
represents hydrophobicity. The hydrophobicity analysis was carried
out according to the Kyte and Doolittle method [22] using
GENETYX
software.
Table 2. Identities of cellular, viral and family members of interleukin
(IL)-10 to the carp gene.
Species
Accession
number
Identities to carp
IL-10 (%)
Overall Mature
Torafugu CAD62446 43.2 45.7
Spotted green pufferfish CAD67773 37.6 39.3
Human NP_000563 28.0 26.6
Cat AAC64705 28.5 26.6
Rat CAA43090 25.2 25.1
Mouse A34853 25.2 25.1
Virus IL-10 homologues
Human Epstein–Barr virus IL-10 CAA24863 27.4 26.9
Equine herpes virus type 2 IL-10 AAB26148 28.0 26.6
Human cytomegalovirus CAA24863 27.0 25.7
Human IL-10 family members

closer to human and pufferfish (torafugu and spotted green
pufferfish) IL-10 sequences. Pufferfish and carp IL-10
genes, clustered together and distant from IL-20 and IL-
24, as recently determined from analysis of the spotted
green pufferfish genome, imply that the carp sequence is
IL-10. The hydropathy analysis also shows similarity of
the torafugu IL-10 sequence to its carp counterpart. These
comparisons suggest that carp IL-10 shares many char-
acteristics of IL-10 with its family members.
The general IL-10 signature sequence comprises a 21
amino acid sequence that is conserved in the carp IL-10
sequence. The first residue in the IL-10 signature sequence is
Lys, except for carp, in which it is Asn. The seventh residue
is His in all three fish sequences, but Gln in mammals. The
second motif (KALGELDL) on the F-helix, which is
conserved in all other family members, is conserved in carp
IL-10. Regions that form the helices in the human IL-10
sequence show a degree of similarity to the carp IL-10
sequence. The most highly conserved stretch in the IL-10
family is at the C-terminus in the C-helix and at the N-
terminus of the F-helix; a similar pattern was also found in
carp IL-10.
By X-ray crystallographic studies, human and viral
homologues of IL-10 are known to have a structure similar
to that of IFN-c, i.e. a noncovalent symmetric homodimer
that forms V-shaped dimers [30,31]. The monomers are
characterized by two disulphide bonds and six a helices. The
structurally important cysteine residues, which form
the disulphide bonds, are all conserved in carp IL-10.
The residues reported to be key in stabilizing the structural

Ó FEBS 2003 Cloning and analysis of IL-10 in fish (Eur. J. Biochem. 270) 4651
The carp IL-10 harbored two instability motifs, in
comparison to five in human IL-10, in the 3¢-UTR.
Whether this difference in the number of instability
motifs affects translation needs to be investigated in
future studies.
LPS induction alone, and costimulation with LPS and
IFN-c or IL-13, resulted in expression of the human IL-10
gene. However, when human monocytes were stimulated
with LPS (100 ngÆmL
)1
) alone, strong expression was not
seen until 2–4 h after stimulation [2]. In our study, we
observed IL-10 gene expression in spleen, head kidney, gill
and intestine from healthy tissues of carp. When stimulated
with LPS, an increase in expression at 1 h poststimulation
was recorded in both head kidney and liver. This indicates
that the gene is inducible by LPS stimulation. Administra-
tion of endotoxin induced IL-10 production in mice,
chimpanzees, baboons and humans [32–35]. The presence
of endogenous IL-10 confers protection from the lethal
effects of endotoxin challenge and reduces the levels of
TNF, IFN-c and macrophage inflammatory protein-2 [36].
Although the expression study indicates that this cytokine is
involved in the immune response, further experiments on
the regulatory mechanisms of carp IL-10 expression and its
role in the regulation of other pro-inflammatory genes, such
as TNF and IFN, in fish, need to be conducted.
In conclusion, the IL-10 gene from carp has been isolated
and its genomic structure and expression analysis investi-

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