Engineering, 2013, 5, 214-219
http://dx.doi.org/10.4236/eng.2013.510B045 Published Online October 2013 (http://www.scirp.org/journal/eng)
Molecular Characterization, 3D Modeling of Grass Carp
Interleukin-10 Receptor 1 (IL10R 1 )
He Wei, Shangnian Wang, Lei Qin, Xinyan Wang, Hong Zhou
School of Life Science and Technology, University of Electronic Science and Technology of China,
Chengdu, China
Received June 2013
ABSTRACT
Interleukin-10 (IL-10) is an important cytokine that plays a pivotal role in natural and adaptive immune systems. How-
ever, in lower vertebrates, especially in teleost the receptor of this cytokine is still largely unknown. This paper de-
scribed the cloning and characterization of grass carp interleukin-10 receptor 1 (gcIL10R1) and the 3D structure of its
extracellular domain was predicted. The gcIL10R1 cDNA included 180 bp 5’ untranslated region (UTR), 870 bp 3’
UTR and an open reading frame (ORF) of 1632 bp. The ORF was found to encode a 543 amino acid protein with a put-
ative JAK1 binding site, one STAT3 binding site. The phylogenetic analysis clusters gcIL10R1 with other teleost
IL10R1s but independently of the amphibian, avian and mammalian IL10R1s. The 3D structure of its extracellular do-
main was the first homology model of a fish IL10R1 that revealed a high similarity with its mammalian and avian
counterparts.
Keywords: Grass Carp; Interleukin-10 Receptor 1; Structure Characterization; 3D Modeling
1. Introduction
Interleukin-10 (IL-10) was first described as an activity
production by mouse Th2 cells that inhibited activation
and cytokine production by Th1 cells [1]. Since its dis-
covery, the biological effects of IL-10 were intensively
investigated in mammals . Known as a cytokine inhibito-
ry factor, the predominant functio n of IL-10 is r egulating
immune responses through direct or indirect effects on
many cell types, including macrophage/monocytes, T
cells, B cells, APCs, and NK cells [2-4]. IL-10 inhibits
the release of pro-inflammatory cytokines like TNF-α,
IL-1β, IL-6, and IL-8 from monocytes/macrophage [5,6].
Moreover, IL-10 inhibits IL-12 synthesis in dentritic
cells [7]. IL-10 also deduces the secretion of IL-23 by
macrophage and IFN-γ production in Th1 cells [8,9]. Our
previous study has identified grass carp IL-10 and inves-
tigated its function in grass carp peripheral blood leuko-
cytes [10].
IL-10 signaling initiates by binding of the ligand to its
specific cell surface receptor. In mammals, the IL-10
receptor complex is composed of two different chains,
IL10R1 and IL10R2. IL10R1 is the ligand-binding sub-
unit and IL10R2 is the accessory subunit which helps
IL-10 signaling transduction. IL10R2 is also part of re-
ceptor complexes of other ligands [11]. IL10R1 was first
be cloned in 1993 [12]. It belongs to the class II cytokine
receptor family whose extracellular domain form fibro-
nectin type III domain that has several conserved amino
acid positions that are important for their secondary
structure [13]. The fibronectin type III domain also exists
in the extracellular region of mammalian IFN-γ receptor
alpha chain [14]. When IL-10 binds to its receptor com-
plex, JAK-STAT signaling pathway is activated and sig-
naling transducer of activation 3 (STAT3) is phosphory-
lated by Janus kinases, JAK1 and Tyk2 [15].
IL-10 genes have been isolated and characterized from
many non-mammalian species, like chicken (Gallus gal-
lus) [16], frog (Xenopus tropicalis) [17], fugu (Fugu ru-
bripes) [18], common carp (Cyprinus carpio L.) [19],
rainbow trout (Oncorhynchus mykiss) [20], zebrafish
(Danio rerio) [21] and grass carp [10] by comparative
genomic analysis or by PCR-mediated homology cloning.
Increasing number of reports described fish IL-10, but
little is known about the IL-10 receptors in teleost. To
date, IL10R1 has only been identified in zebrafish and
goldfish [22] by gene synteny ana lysis.
In this investigation, IL10R1 cDNA has been identi-
fied and characterized from grass carp. The 3D model of
extracellular domain of gcIL10R1 protein was predicted,
representing the first 3D analysis of the quaternary
structure of a fish IL10R1, which provides the basis for
further investigation of the IL-10 signaling in teleo s t.

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215
2. Material and Methods
2.1. Animals
The 1-year-old Chinese grass carp (Ctenopharyngodon
idellus) with 1 - 1.5 kg body weigh were obtained from
Chengdu Tongwei Aquatic Science and Technology
Company. An acclimation period of two weeks was ob-
served prior to the experiments commencing. Tissues for
cloning of IL-10R2 were taken from freshly killed fish
according to the Regulation of Animal Use in Sichuan
province, China.
2.2. RNA Extraction and cDNA Synthesis
Total RNA was extracted from grass carp spleen with
Tripure Reagent (Roche, Basel, Switzerlan) according to
the manufacturer’s instructions. After extraction of total
RNA, five μg was reverse transcribed to cDNA using
Superscript II reverse transcriptase (Invitrogen, Carlsbad,
CA) with oligo d(T)18 pr ime r .
2.3. Cloning of Grass Carp IL10R1
Partial cDNA sequence of the grass carp IL10R1 gene
was obtained by PCR using the primers IL10R1F and
IL10R1R (Table 1), which was designed basing on the
conserved regions of goldfish and zebrafish IL10R1s.
PCR was performed with one cycle of incubation at 94˚C
for 3 min, followed by 35 cycles at 94˚C for 30 s, 58˚C
for 30 s and 72˚C f or 1min with a final cycle at 72˚C for
10 min. To obtain the complete grass carp IL10R1 se-
quence, 5’ and 3’ RACE techniques were used to obtain
the 5’ and 3’-end sequences of grass carp IL1 0R1 cDN A.
Referring to the nucleotide sequence of the partial se-
quenced grass carp IL10R1 cDNA, gene-specific primers
were designed for 3’ and 5’-RACE (Table 1), and PCR
was performed with a GeneRacer Kit (Invitrogen) ac-
cording to the protocol.
2.4. Sequence Analysis and Phylogeny Analysis
The full cDNA sequence and deduced amino acid se-
quence of IL-10 were analyzed using BLAST program
from NCBI and the ExPASy Molecular Biology server
Table 1. Oligonucleotide primers used to amplify grass carp
IL10R1.
Name Sequence (5’to 3’) object
IL10R1F ATATGGGAGGGAATGTAACTG
partial
IL10R1R TCCTGGTTTTGCACCACATGC
IL10R1-5N1 TCACCATCTTATCATCATCATC
5’-race
IL10R1-5N2 CTTTCTGTGAATTTTAACCTTC
IL10R1-3N1 TCAGCAGTGGAAGAAGAAGC
3’-race
IL10R1-3N2 GATGGATATCGTAGTCAGAG
(http://us.expasy.org). The multiple alighments were
made using DNAMAN software (Lynnon Biosoft, Pointe-
Claire, Canada). The molecular mass and isoelectric
point of the putative grass carp IL10R1 were predicted
using Compute PI/Mw tool at the ExPASy
(http://www.expasy.c-h/). The deduced signal peptide
was predicted using SignalP [23] and the transmembrane
regions were predicted using the TMHMM Server v. 2.0
(http://www.cbs.dtu.dk/ser vices/T-HMM/). The fibro-
nectin type III domain and the immunoglobulin-like fold
of the grass carp IL10R1 were predicted using the Inter-
Pro Scan online server
(http://www.ebi.ac.uk/Too ls /pfa/ip-rscan/). The putative
N-glycosylation sites of the grass carp IL10R1 were de-
termined using the NetNGlyc Server v. 1.0
(http://www.cbs.dtu.dk/-services/netnglyc/). Phylogenetic
trees were constructed using MEGA3.1 software [24].
The genetic distance between species was calculated us-
ing ρ−distance method. The cladogram was generated
using the neighbor-joining (NJ) method. In the analysis,
the gaps were deleted, and a 1000 bootstrap procedure
was used to test the robustness of the node on the trees.
2.5. 3D Modeling
Two suitable structural templates for grass carp IL10R1
extracellular domain, chicken IFN-γ receptor alpha chain
extracellular domai n s and human IFN-γ receptor alpha
chain extracellular domain were identified by a BLAST
search as implemented in the SWISS-MODEL Protein
Modelling Server (http://www.isb-sib.ch/ ). The automat-
ic sequenc-e alignment thus obtained was used for ho-
mology modeling with SWISS-MODEL. The resulting
theoretical model of a protein monomer was displayed
and analyzed with Swiss-PDB Viewer. Models pictures
were obtained using the PyMOL program
(http://www.pymol.sour c efor-ge.net/index.html).
3. Results
3.1. Molecular Cloning and Sequence Analysis
of Grass Carp IL10R1
By using the primers which were designed on the basis
of the conserved region of the known fish IL10R1 se-
quences and 5’ and 3’ RACE, we got the cDNA se-
quences of grass carp IL10R1 (Figure 1). The gcIL-
10R1 shared 73% homology with amino acid sequence
of goldfish IL10R1, and gcIL10R1 protein had conserved
four cysteine residues as well as partially conserved hy-
drophobic residues, suggesting importance in the st ruc-
tural integrity and receptor-ligand interactions. The
gcIL10R1 protein had a putative fibronectin type III do-
main, an immunoglobin-like fold and several predicted
N-glycosylation sited. Its sequence also bared putative

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216
Figure 1. Nucleotide and deduced amino acid sequences of
grass carp IL10R1.
JAK1 (SVLLFKK) and STAT3 (YXXQ) binding sites.
The identified gcIL10R1 possessed one STAT3 binding
site (Figure 2). Furthermore, like other fish, the C-ter-
minal serine-rich areas were present in gcIL10R1 protein,
which proposed to be essential for the immunosuppres-
sive effects of the mammalian IL10R1 [25].
Phylogenetic analysis of gcIL10R1 proteins demon-
strated their close relationship with the zebrafish and
goldfish IL10R1s. The IL10R1 proteins of the abov e fish
species branched independently to the IL10R1s of other
vertebrates, while the amphibian and avian IL10R 1s also
branched independently from the IL10R1 of mammals
(Figure 3).
3.2. 3D Modeling
The structure of gcIL10R1 extracellular domain which
was for ligand binding was predicted by comparative
modeling using the extracellular domains of chicken
IFN-γ receptor alpha chain (PDB id: 4EQ3) and human
IFN-γ receptor alph a chain (PDB id: 3S8W) as template.
The homology model of grass carp IL10R1 has thr ee-
helix and several beta strands (Figure 4). The alignment
used in the model shows a high similarity (75%) between
the target molecular and comparing extracellular do-
mains of human and chicken IFN-γ receptor alpha chain.
4. Discussion
In the present study, IL10R1 cDNA was cloned and cha-
racterized. For gcIL10R1, an open reading frame of 1632
bp was found to encode a 543 amino acid protein. The
putative grass carp IL10R1 is a 60016 molecular mass
polypeptide with a pI of 4.5. The gcIL10R1 shares high
homology with amino acid sequence of other known te-
leost IL10R1. Analysis of gcIL10R1 sequence revealed
the conserved putative JAK1 and STAT3 binding sites.
JAK1 binding sites on the mammalian IL10R1 were
identified in a region consisting of four core hydrophobic
residues [26,27], comprising of the SVLLFKK motif on
the human IL10R1. Our analysis of gcIL10R1 revealed
that this region has been partially conserved evolutiona-
rily, with gcIL10R1 proteins exhibiting the AVLKSAV
motif in the corresponding region. The STAT3 binding
motif (YXXQ), present on the mammalian IL10R1 is
indispensable to IL-10 function [28,29]. This motif ap-
pears to have been evolutionarily conserved across ver-
tebrate IL10R1 proteins including teleost. Ho wev er,
while the amphibian, avian and mammalian IL10R1 pro-
teins possess two potential STAT3 binding sites, the
grass carp and other teleost IL10R1 proteins appear to
only have one putative STAT3 docking site. It will be
interesting to learn the significance of having one rather
than two STAT3 binding sites.
The predicted 3D structure for the gcIL10R1 extracel-
lular domain revealed a high similarity with IFN-γ re-
ceptor alpha chain. Since IL-10 has similar structure with
IFN-γ [14], our results indicated that gcIL10R1 is suita-
ble for binding with grass carp IL-10.
Our studies cloned and analy zed the gcIL10R1 and its
3D structure was modeled. Those results suggested
gcIL10R1 is structurally similar to its counterpart in
mammals or avian, which pave the way for further inves-
tigation on IL-1 0 s ignaling in fish.

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Figure 2. Alignment of the amino acid sequence of grass carp IL10R1 with other homologues. The conserved cysteine resi-
dues are denoted by *. Conserved hy drophobi c residues are denoted by arr ows. The pr edicted gly cosylati on sites are denot ed
by overhead Y. The IL10R1 fibronectin type III domain and immunoglobin fold are indicated by an overhead line. Potential
JAK1 and STAT binding site s are boxed.

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Figure 3. Phylogenetic analysis of the IL10R1. An unrooted
phylogenetic tree constructed by the neighbor joining me-
thod using MEGA software from the amino acid sequences
of IL10R1s. Grass carp IL-10R1 was boxed. The numbers
indicate the bootstrap confidence values obtained for each
node after 1000 replications. The accession numbers of the
sequences used in the alignment and in Figure 2 are as fol-
lows: Human IL10R1:NP_001549, Monkey IL10R1: XP_
001092736, Pig IL10R1: XP_003129938, Mouse IL10R1:
NP_032374, Frog IL10R1: AAI59332, Chicken IL10R1:
NP_001034686, Turkey IL10R1:XP_003212786, Goldfish
IL10R1: JN203498, Zebrafish IL10R1: NP_001071093,
Grass carp: KF129394.
Figure 4. Homology modeling of grass carp IL10R1. Yellow
arrows represent beta strands, red arrows represent alpha
helices.
5. Acknowledgements
This work was supported by grant from the National
Natural Science Foundation of China (31101877).
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