Molecular characterization and expression analysis of the putative interleukin 6 receptor (IL-6Rα and
glycoprotein-130) in rainbow trout (Oncorhynchus mykiss): salmonid IL-6Rα possesses a polymorphic
N-terminal Ig-domain with variable numbers of two repeats
Maria M. Costa1,2, Tiehui Wang1, Milena M. Monte1 and Christopher J. Secombes1*.
1
Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen AB24 2TZ, UK.
Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC),
Eduardo Cabello 6, Vigo, Spain.
2
*
Author for correspondence:
Prof. Christopher J. Secombes
Scottish Fish Immunology Research Centre,
School of Biological Sciences,
University of Aberdeen,
Aberdeen AB24 2TZ, UK.
Tel:0044-1224-272872
Fax:0044-1224-272396
Email: c.secombes@abdn.ac.uk
The nucleotide sequence data will appear in the EMBL/DDBJ/GenBank nucleotide sequence database under
the following accession numbers: FN824530 (IL-6Rα) and FN824531 (gp130).
1
Abstract
Interleukin (IL)-6, the founding member of IL-6 family cytokines, plays non-redundant roles in hematopoiesis
and acute phase responses. IL-6 signals via a specific private IL-6Rα and a common beta chain gp130. In this
study, we sequence analysed both IL-6Rα and gp130 in rainbow trout. The trout gp130 cDNA encodes 906 aa
and is similar in size, extracellular domain structure (D1-6) and presence of intracellular motifs important for
signal transduction to tetrapod gp130. The trout IL-6Rα cDNA encodes for 834 aa and is larger compared to
tetrapod IL-6Rαs, as are other fish IL-6Rα molecules due to a large D1 domain. However, the cytokine
binding domain is well conserved across vertebrates, with four conserved cysteine residues in the N-terminal
FNIII domain and a WSXWS motif in the C-terminal FNIII domain. Furthermore, phylogenetic tree analysis
confirmed that the reported fish IL-6Rα and gp130 molecules are orthologues to their tetrapod counterparts.
The extra-large D1 domain of the salmonid IL-6Rα molecules results from the insertions of two repetitive
sequences of [TS]-[TF]-VSTTT-[ND]-TTSNG and TTVS-[AT]-IKD-[DG]-S-[KD]-N-[GR], respectively.
Furthermore the numbers of repetitions of the two motifs were variable in different individuals and cell lines,
and even in the same fish allelic polymorphism exists. Trout IL-6Rα was expressed at higher levels than
gp130 in a number of tissues examined and the expression of both IL-6Rα and gp130 could be modulated by
LPS and Poly I:C in four trout cell lines studied. The expression patterns of the receptors suggest that high
level expression of IL-6Rα is critical for IL-6 responsiveness.
2
3
Introduction
Interleukin (IL)-6 is the founding cytokine of the IL-6 family that comprises in addition to IL-6, IL-11, IL-27,
leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), CT-1, CT-2 and
cardiotrophin-like cytokine (CLC) and neuropoietin (NPN) (Huising et al. 2005; Wang et al. 2005; Silver and
Hunter 2010; Hirano 2010). Generally, they are involved in inflammatory and immunologic processes as well
as in hematopoiesis, liver and neuronal regeneration, embryonic development and cardiovascular physiology.
Through activation of target genes involved in growth, differentiation, survival, apoptosis, and proliferation,
IL-6 family cytokines play a key role in cellular and tissue homeostasis (Fischer and Hilfiker-Kleiner 2007).
In mammals, all the IL-6 family cytokines signal via a common glycoprotein, known as gp130, regardless of
the contribution played by the cytokine-specific α chains, e.g. IL-6Rα (Gerez et al. 2007). Engagement of
gp130 with cytokine-specific α chains triggers the activation of at least three different downstream signalling
pathways, the signal transducers and activators of transcription (STAT) 1 and 3, the Src-homology tyrosine
phosphatase 2 (SHP2)-Ras-ERK, and the PI3K-Akt system (Silver and Hunter 2010; Hirano 2010). Due to the
common use of gp130 as a signal chain, the IL-6 family cytokines are highly redundant in their capacity to
mediate biological functions. They also show member specific activity. For example, IL-6 deficient mice
exhibit defects in hematopoiesis, acute phase protein synthesis, antigen-specific antibody production,
chemokine induction, leukocyte recruitment, hepatocyte regeneration and susceptibility to infections
(Fasnacht and Müller 2008).
IL-6 can induce cell signalling not only via the classic pathway involving the transmembrane receptor IL-6Rα
associated with gp130, but also via the soluble receptor IL-6Rα, which binds to IL-6 and induces a signal
mediated by the ubiquitous gp130 molecule (trans-signalling) (Assier et al. 2010). This second pathway
widens the scope of IL-6 signalling since cells expressing no membrane bound IL-6R can also be stimulated
by the trans-signal pathway. Both IL-6Rα and gp130 are type-I cytokine receptors, also known as
hematopoietin receptors, that share a modular architecture with an N-terminal extracellular and C-terminal
intracellular orientation. Every type I cytokine receptor has a characteristic ~200 aa cytokine binding
homology domain (CBD) consisting of two fibronectin type-III (FNIII) domains from the N-terminal to Cterminal. The N-terminal domain contains four conserved cysteine residues that form two intrachain disulfide
bonds, whilst the C-terminal domain has a conserved WSXWS motif that has been proposed to be required for
maintaining the receptor in a conformation favourable to cytokine binding (Bazan 1990; Boulay et al. 2003).
In addition, both receptors have an immunoglobulin (Ig)-like domain N-terminal to the CBD and gp130, that
belongs to the class of “tall cytokine receptors”, also has three FNIII-like domains between the CBD and the
transmembrane domain (Waetzig et al. 2010). Thus, IL-6Rα and gp130 have 3 (D1-3) and 6 (D1-D6) domains
within their extracellular regions, respectively.
4
The type I cytokine receptors have no intrinsic enzyme activity but are constitutively associated with tyrosine
kinases of the Janus kinase (JAK) family through the box 1 and/or box 2 motifs in the intracellular region of
the signalling chains. Cytokine binding induces receptor oligomerization and activation of associated JAKs
that phosphorylate themselves and the intracellular domains of the receptors. The phosphorylated tyrosine
residues in the receptors then serve as docking sites for a second family of proteins, the STAT molecules.
Binding of STATs to intracellular domains of receptors leads to their tyrosine phosphorylation and subsequent
dissociation from the receptors. The phosphorylated STATs form dimers and translocate into the nucleus,
where they bind to specific regulatory elements in the promoters of responsive genes and act as transcription
factors to modulate gene expression (Murray 2007; Hershey 2003). The intracellular domain of IL-6Rα lacks
STAT binding sites and is devoid of signalling (Silver and Hunter 2010). Mammalian gp130 possesses
conserved boxes (1-3), and multiple docking sites, mainly for STAT3 but also STAT1 and STAT5, in the
intracellular domain (Heinrinch et al. 1998).
Gp130 is expressed ubiquitously. Therefore it has so far been a generally accepted idea that the
responsiveness of individual cell types to IL-6-type cytokines is mainly determined by the expression of a
specific α receptor (i.e. IL-6Rα) or by the expression of a second specific signal-transducing subunit. It is
known that mammalian IL-6Rα expression is limited to particular cell types such as neutrophils,
monocytes/macrophages and some lymphocytes (Waetzig et al. 2010).
A few IL-6 family members have been successfully identified recently in several fish species. The orthologues
of mammalian IL-6 have been cloned in fugu (Tagifugu rubripes) (Bird et al. 2005), rainbow trout
(Oncorhynchus mykiss) (Iliev et al. 2007), flounder (Paralichthys olivaceus) (Nam et al. 2007) and seabream
(Sparus aurata) (Castellana et al. 2008), as well as IL-11 in rainbow trout (Wang et al. 2005), carp (Huising et
al. 2005) and flounder (Santos et al. 2008). In addition, novel fish IL-6 family members have also been
discovered including a fish IL-11 like molecule (Huising et al. 2005; Santos et al. 2008), the M17 molecule
that has homology to mammalian LIF/OSM (Fujiki et al. 2003; Hwang et al. 2007; Wang and Secombes
2009) and a CNTF-like gene, that may be homologous to an ancestral gene that gave rise to mammalian
CNTF/CLC/CT-1/CT-2 (Wang and Secombes 2009). The functional characterisation of these fish molecules
has been started. For example, goldfish M17 induces differentiation of and nitric oxide production by
macrophages (Hanington and Belosevic 2007) and trout IL-6 promotes macrophage growth and induces
antimicrobial peptide gene expression (Costa et al. 2011).
The characterisation of the receptor subunits of fish IL-6 family members is scarce (Hannington and Belosevic
2005; Santos et al. 2007), despite the large number of putative receptors that have been annotated after the
sequencing of several model fish genomes (Aparicio et al. 2002; Jaillon et al. 2004; Liongue and Ward 2007)
and various large expressed sequence tag (EST) projects (Leong et al. 2010). In the current paper, we have
sequence analysed IL-6Rα and gp130 in rainbow trout, and compared them with other predicted fish
5
homologues. We found that the fish IL-6Rα possesses a large Ig-like D1 domain and in salmonids this D1domain was further enlarged by an insertion of two types of 13 aa repeat sequences that exist at different
frequencies. We examined the expression of the two receptors in healthy fish and found that the expression of
trout IL-6Rα was generally higher than that of gp130. We further analysed their expression and modulation in
four trout cell lines after stimulation with lipopolysaccharide (LPS) and polyinosinic: polycytidylic acid (Poly
I:C), as two common pathogen-associated molecular patterns (PAMPs).
6
Materials and methods
Fish
Rainbow trout were obtained from a commercial Scottish fish farm (Almond bank. Perthshire, UK). The
animals were maintained in recirculating filtered freshwater tanks (1 m diameter fibreglass/350 L) at 15 ± 1°C
with aeration. They were fed twice daily with a standard commercial trout diet (Ewos, Scotland). Prior to
experimental work, animals were acclimatized to laboratory conditions for a minimum period of two weeks.
Cell lines
The rainbow trout monocyte/macrophage cell line RTS-11 (derived from spleen cells, Ganassin and Bols
1998) was maintained in Leibovitz medium (L-15; Invitrogen) containing 30% foetal calf serum (FCS;
Labtech International) and antibiotics (Penicillin-Streptomycin (P/S), Invitrogen) at 20°C. The cell lines,
RTG-2 (a fibroblast-like cell line derived from gonad; Wolf and Quimby 1962), RTGill (Gill-derived;
Schirmer et al. 1998) and RTL (an epithelial cell line derived from liver; Lee et al. 1993), were maintained in
the same medium as for RTS-11 cells except with reduced FCS (10%) as described previously (Wang et al.
2011b). Cells were maintained in 75 cm2 flasks at 20 °C until confluent and then sub-cultured into new media.
Cloning of IL-6Rα and gp130
Two Atlantic salmon Salmo salar IL-6Rα like sequences (ACN10857 and ACN11013) were found in the
NCBI database with multiple numbers of two repeats within the Ig-like domain that is crucial for ligand
receptor binding. To verify if these repeats also exist in other salmonids we sequence analyzed the IL-6Rα in
rainbow trout. Blast search of the EST database at NCBI (http://blast.ncbi.nlm.nih.gov/Blast) using the salmon
sequences identified multiple trout ESTs encoding the C-terminal of a potential trout IL-6Rα homologue.
Primers F1 and F2 (Table 1) were designed against the ESTs BX883008 and GE837037 and used for 3’RACE using SMART cDNA prepared from head kidney (HK) as described previously (Wang et al. 2008). A
1 Kb 3’-RACE product was obtained that contained the sequence encoding for the C-terminal and the 3’untranslated region (UTR) of the trout IL-6Rα. A 2.8 Kb product was amplified using primers F3/R3 (Table
1) designed to the 5’- and 3’-UTR and that contained the complete coding region of trout IL-6Rα. The primer
positions are marked in supplementary Figure 1.
Blast search of the NCBI EST database using the mammalian gp130 protein sequence identified multiple
salmonid ESTs that were potentially derived from a salmonid gp130. To obtain the complete coding region,
primers F1 and R1 (Table 1 and supplementary Figure 2) were designed to the 5’- and 3’-UTR of ESTs
7
EG758643 and CU065376, respectively, and used for PCR. A 3 Kb band was amplified from a HK cDNA
sample that contained the complete coding region of gp130.
Analysis of the frequency polymorphism of the two repeats in IL-6Rα
The expression of IL-6Rα was detected in different cell lines and tissues at different levels. To ensure the
amplified bands of the repeated cDNA sequences could be revealed on an agarose gel, two rounds of PCR
were performed. The first PCR was carried out using primer pair repF1/R1 (Table 1 and supplementary Figure
1) and the resultant product was diluted 100 fold and used as template for the second nested PCR using primer
pair repF2/R2 (Table 1). The amplified products were run on a 1% agarose gel containing ethidium bromide
and selected PCR products were cloned and sequence analyzed as above.
Sequence and phylogenetic analysis
The obtained nucleotide sequences were analyzed and assembled using the AlignIR program (LI-COR). A
multiple sequence alignment was generated using ClustalW2 (Chenna et al. 2003) and the translation was
performed using ExPaSy tools (http://us.expasy.org/tools). The domain distribution was analysed with Simple
Modular
Architecture
Research
Tool
(SMART,
http://smart.embl-heidelberg.de/),
Scan
Prosite
(http://expasy.org/prosite/) (Gasteiger et al. 2005) and Pfam (http://pfam.sanger.ac.uk/) (Finn et al. 2010). The
signal peptide was predicted used SignalP 3.0 (Emanuelsson et al. 2007) and secondary structure predicted
using
SOPM
(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopm.html),
YASPIN
http://www.ibi.vu.nl/programs/yaspinwww/) and homology with other species. Phylogenetic trees were
created by the Maximum Likelihood method based on the JTT matrix-based model using the MEGA program
(version 5) (Tamura et al. 2011), and were bootstrapped 2,000 times.
Tissue distribution
Tail fins, gills, thymus, brain, scales, skin, muscle, liver, spleen, gonad, head and caudal kidney, intestine and
heart from six healthy animals were harvested in order to analyze the expression level in different tissues.
Samples were homogenised and kept in TRI reagent (Sigma) until their processing.
RNA isolation, cDNA production and real-time PCR analysis of gene expression
RNA isolation from TRI lysate was performed following the manufacturer’s protocol. 5-10 µg total RNA was
converted into cDNA using Bioscript (Bioline). The cDNA generated was diluted with 400 μl of TE buffer
(10 mM Tris HCl; 1 mM EDTA, pH 7.5) and 4 μl were used as template for quantitative PCRs using the
primers described in Table 1. Real time PCR analysis was performed as previously (Wang et al. 2007; 2011),
using a Light Cycler real-time PCR system (Roche). The expression of the candidate genes was normalized to
8
the housekeeping gene EF-1α. Fold change was calculated by dividing the normalized expression values of
stimulated cells by that of the controls.
Modulation of gene expression
of cells (5 ml at 5×105 cells/ml) were seeded into 25 cm2 cell culture flasks and incubated at 20°C overnight.
The cells were then stimulated with E. coli LPS (Sigma-Aldrich) and Poly I:C (Sigma-Aldrich) at a final
concentration of 25 µg/ml and 50 µg/ml, respectively. The stimulation was terminated at 4, 8 and 12 h by
dissolving the cells in TRI reagent.
Statistics
The SPSS package 17.0 (SPSS Inc. Chicago, Illinois) was used for statistic analysis of changes of gene
expression levels. To improve the normality of real-time quantitative PCR measurements, a log2
transformation of the arbitrary units after normalization to the expression level of EF-1α was performed as
described previously (Wang et al. 2011a; b). The relative expression levels of IL-6Rα and gp130 in tissues
were analysed by paired samples T tests. One way-ANOVA and the LSD post hoc test was used to analyze
the expression data in the cell lines after stimulation, with P <0.05 between treatment groups and control
groups considered significant.
9
Results
Sequence analysis of rainbow trout IL-6Rα
The cDNA sequence of the rainbow trout IL-6Rα chain (rtIL-6Rα), isolated from a HK cDNA sample (EMBL
acc. no. FN824530), was 2956 nucleotides with a 5’-UTR of 182 bp, a long open reading frame (ORF)
encoding for 834 aa and a 3’-UTR of 244 bp followed by a poly A tail (Supplementary Fig. 1). There were
four upstream ATGs before the main ORF in the 5’-UTR and a polyadenylation signal (AATAAA) 13 bp
upstream of the poly A tail in the 3’-UTR. The translation from the main ORF had a predicted signal peptide
of 19 aa, an extracellular region of 707 aa, a transmembrane domain of 23 aa and an intracellular region of 85
aa. The extracellular region of mammalian IL-6Rα contains an immunoglobulin-like (Ig-like) domain (D1),
and a cytokine binding domain (CBD) composed of two FNIII domains (D2 and D3) that each contain seven
β-strands. Whilst the D2 and D3 of CBD were identifiable in trout IL-6Rα and were conserved among IL-6Rα
molecules from mammals, birds and fish, the trout Ig-like D1 domain was relatively large as also seen in
salmon IL-6Rα molecules. This was due to the presence of multiple copies of two different motifs
corresponding to the 13 aa peptide sequences [TS]-[TF]-VSTTT-[ND]-TTSNG and TTVS-[AT]-IKD-[DG]S-[KD]-N-[GR] respectively. Eight repetitions of the first motif and 7 of the second were found in the
translation of the cDNA sequence from HK (Supplementary Fig. 1). Multiple N-glycosylation sites (NXT/S)
were also present in the extracellular region of the translation, with each repeated motif containing a potential
N-glycosylation site.
The trout IL-6Rα shared 84% aa identity to the two Atlantic salmon IL-6Rα EST entries, and even higher
identities (90.3% and 90.9%) when the amino acid repeat sequence was removed (Table 2). It shared 27.5%
and 24.4% identities to IL-6Rα molecules from tetraodon and zebrafish, respectively, but only marginal (16.118.9%) identities to tetrapod IL-6Rα molecules. In contrast, the tetraodon and zebrafish IL-6Rα molecules
shared higher identities (23.0-26.8%) to their tetrapod orthologues. However, when the aa repeat sequences in
the salmonid IL-6Rα molecules were removed for analysis, comparable identities (21.0-24.7%) to tetrapod
orthologues were seen as with tetraodon and zebrafish (Table 2). It is worth noting that the fish IL-6Rα
molecules shared the highest identities to tetrapod IL-6Rα relative to other IL-6Rα related tetrapod sequences
including IL-11Rα and CNTFR (Table 2).
To compare the aa sequence of salmonid IL-6Rα and their orthologues from other species, a multiple
alignment was constructed using IL-6Rα molecules from tetraodon and representative tetrapods. Only one
Atlantic salmon sequence (acc. no. ACN10857) was used as the two salmon sequences shared 96.1% identity
when the sequence repeats were removed (Table 2). The zebrafish IL-6Rα sequence was also excluded as the
N-terminal of this sequence was likely incomplete. The alignment showed that the extracellular D2 and D3
domains, and to a lesser extent the D1 domain, are relatively conserved except for an insertion in the D1
10
domain in fish IL-6Rα molecules (Table 2). Thus, all the fish IL-6Rα molecules are larger than the tetrapod
ones. The salmonid IL-6Rα molecules were further enlarged by the two aa repeat sequences (Fig. 1). The
conserved features included 10 cysteine residues (C1-10), a proline rich hinge region between the D1-D2 and
D2-3 domains, and the type I cytokine receptor signature (WSXWS). Of the 10 conserved cysteine residues, 8
of them (C1-5, 7, 9 and 10) are completely conserved across vertebrates, whilst C6 and C8 were conserved
only in fish (Fig.1). It is known that C2-3 in D1, C4-5 in D2 and C7-9 in D2 form disulphide bonds in the
human IL-6Rα (Cole et al. 1999). In addition, the N-terminal D1 is disulphide-bonded to D2 through C1 and
C10 (Varghese et al. 2002). The conserved C6 and C8 in fish IL-6Rα may form an additional disulphide bond
to stabilise their tertiary structure. Each IL-6Rα molecule from different species has multiple putative Nglycosylation sites, but none of them are completely conserved across all vertebrates.
Sequence analysis of rainbow trout gp130
A 2,968 bp cDNA fragment was amplified using primers F1 and R1 that contained a 5’-UTR of 169 bp, an
ORF of 2721 bp, and part of the 3’-UTR of the trout gp130 gene (Supplementary Fig. 2, EMBL acc. no.
FN824531). There were three upstream ATGs before the main ORF that could be potentially translated into
three short peptides, and at least 6 mRNA instability motifs (ATTTA) in the 3’-UTR. The main ORF encodes
for a 906 aa protein with a predicted signal peptide of 26 aa, an extracellular region of 609 aa, a
transmembrane domain of 21 aa and an intracellular region of 250 aa (Supplementary Fig. 2). Structurally,
trout gp130 is predicted to be composed of one C2-type Ig-like domain (D1) and five FNIII domains (D2-D6)
in its extracellular portion. As with other type I cytokine receptors, rtgp130 possesses a WSXWS motif in the
D3 domain (Supplementary Fig. 2). The trout translation had 14 putative glycosylation sites in the
extracellular domain.
The trout gp130 had a similar size (906 aa) to tetrapod gp130 molecules but shared higher aa identities of
42.5% (zebrafish) and 40.7% (tetraodon) to fish gp130s relative to tetrapod gp130 molecules (33.7-35.7%,
Table 3). The gp130 molecules from fish, amphibians and birds shared highest aa identities to mammalian
gp130 compared to any related mammalian sequences including GCSFR, LIFR, OSMR and IL-31Rα. It is
worth noting that mammalian gp130 molecules share high aa identities (76.2-90.8%), whilst only 38.5-42.5%
identities were shared by fish gp130 molecules. The amphibian and bird gp130 also had low identities to
gp130 molecules from fish and mammals (Table 3).
To compare the aa sequence of gp130 from trout and other species, a multiple alignment was constructed
using all the known gp130 molecules from fish and selected tetrapod gp130 molecules. Good conservation
was seen across the whole molecule, including a signal peptide, a six domain (D1-D6) extracellular region, a
transmembrane domain and an intracellular domain. There were 4 conserved cysteine residues in the
extracellular region, with an additional 7 conserved in tetrapods (Fig. 2). In human gp130 C1-C3 and C2-C4
in the D1 domain, C5-C6 and C7-C8 in the D2 domain, and C9-C10 in the D5 domain form disulphide bonds
11
(Moritz et al. 2001). Other conserved extracellular features were proline rich hinge regions between the (D16) domains, the type I cytokine receptor signature (WSXWS) in the D3 domain, and two N-glycosylation
sites, one in the middle of the D1 domain and the other at the N-terminal of D2.
As the signal chain of the IL-6R, gp130 contains several conserved critical intracellular regions named boxes
1-3 (Murakami et al. 1991; Baumann et al. 1994; Lai et al. 1995) that are required for different signalling
mechanisms. All the three boxes were well conserved across vertebrates (Fig. 2). The YXXQ motif in the
intracellular region of mammalian gp130 is a known docking site for STAT3, and critical for the
phosphorylation of STAT3 on Ser727 in human gp130 (Abe et al. 2001). Four YXXQ motifs (YXXQ1-4) are
also conserved in tetrapod gp130, and YXXQ1 and 3 are also conserved in fish gp130 with YXXQ4 also
conserved in some fish but not trout (Fig. 2). The YSTV motif, a known docking site for SHP-2 and important
for activation of the Ras/Raf/MEK/Erk pathway, is also conserved across vertebrates with YSTV in tetrapods
and YSSV in fish (Fig. 2). The cell surface expression of gp130 is tightly regulated by internalization via a dileucine dependent internalization motif (Radtke et al. 2010). The internalization motif, as well as a serine rich
region, were well conserved in gp130 molecules across vertebrates.
Phylogenetic tree analysis
To confirm the identities of trout IL-6Rα and gp130, un-rooted Maximum Likelihood (ML) trees (Fig. 3) were
inferred using MEGA5 software (Tamura et al. 2011) with the Jones-Thornton-Taylor (JTT) aa matrix and all
sites. In the IL-6 receptor alpha chain tree, the known fish and selected tetrapod IL-6Rα, CNTFR and IL-11Rα
were included in the analysis. The fish IL-6Rα molecules formed an independent clade and grouped with IL6Rα molecules from tetrapods with high bootstrap support (100%). They were separate from the CNTFR and
IL-11Rα groups, further supporting the identities of fish IL-6Rα molecules (Fig. 3A).
In the gp130 and related gene phylogenetic tree analysis, all the known gp130 molecules from fish and from
selected tetrapods, as well as mammalian molecules closely related to gp130, including GCSFR, LIFR,
OSMR and IL-31Rα, were analysed. All the related receptors formed independent clades with high bootstrap
support (100%) and were separate from the gp130 group. The fish gp130 molecules grouped within the gp130
clade with good bootstrap support (80%) (Fig. 3B).
Repeat polymorphism
Using nested PCR with primers covering the whole repeat region in the Ig-like D1 domain of the IL-6Rα
chain, amplicons with similar size could be amplified from all the 14 tissues examined from the same fish
(Fig. 4A). However, the size of the amplicons could vary between fish, and it was possible to obtain more than
one. Furthermore, the amplicons from 4 different cell lines showed great differences in size, from 0.7 Kb to
1.3 Kb (Fig. 4B), probably because they originated from different fish. To further investigate the nature of the
12
repeat polymorphism, amplicons from different fish and cell lines were cloned and sequence analyzed. We
found that individual fish or cell lines could possess more than one pattern of repeats, as shown in Figure 4C.
The numbers of repeats found in the analysed samples varied between 3 to 13 for the first 13 aa repeat and
between 4 to 17 for the second repeat. Since the repeats were associated with potential glycosylation sites, this
meant that the trout IL-6Rα from different individuals may also have different N-glycosylation potential.
Tissue distribution of expression of IL-6Rα and gp130
The expression of IL-6Rα and gp130 was detectable by real-time PCR in all the 14 tissues examined. The
lowest transcript expression level of IL-6Rα was in the scales (4 units), whilst for gp130 it was in the intestine
(1 unit, Fig. 5A). The highest expression of IL-6Rα was in spleen (448 units), followed by head kidney, liver,
and caudal kidney. IL-6Rα was also moderately expressed in gills, brain, heart, thymus and intestine (20-55
units). The highest expression of gp130 was in muscle (60 units), followed by brain, gonad and skin. Gp130
expression in other tissues was low (1-9 units, Fig. 5A). In general, the expression level of IL-6Rα was higher
than that of gp130 in most of the tissues examined, except in brain, scales, skin, muscle and gonad where the
expression of IL-6Rα and gp130 was not significantly different (Fig. 5B). For example, the expression of IL6Rα was 102, 77, 51 and 37 fold higher than that of gp130 in head kidney, liver, spleen and gills, respectively
(Fig. 5B).
Modulation of IL-6Rα and gp130 expression in four trout cell lines
Both IL-6Rα and gp130 were highly expressed in the monocyte/macrophage cell line RTS-11, at comparable
levels (Fig. 6A). The epithelial RTL cell line and fibroid cell lines RTG-2 and RTGill also expressed moderate
levels of gp130 but only expressed low levels of IL-6Rα (Fig. 6A).
The expression of IL-6Rα and gp130 could be modulated to some degree in a cell type and time dependent
manner by LPS and Poly I:C (Fig. 6B and 6C). A moderate increase of IL-6Rα expression was seen at 8 h and
24 h in RTL and RTG-2 cells, and at 24 h in RTS-11 cells after LPS stimulation. However, IL-6Rα expression
was decreased at 4 h in RTL and at 24 h in RTGill after LPS stimulation (Fig. 6B). IL-6Rα expression was
refractory to Poly I:C stimulation in RTG-2, RTGill and RTS-11 cells, but showed a small increase at 8 h in
RTL. In contrast, gp130 expression was refractory to LPS stimulation in RTL, RTG-2 and RTGill cells, whilst
in RTS-11 cells the high level constitutive expression of gp130 was down regulated at 8 h by LPS (Fig. 6C).
Poly I:C was able to increase gp130 expression at 8 h and 24 h in RTG-2 and at 4 h and 8 h in RTGill, and
like LPS it decreased gp130 expression at 8 h in RTS-11 cells (Fig. 6C).
13
Discussion
In this report, the rainbow trout IL-6Rα and gp130 were cloned and sequence analysed. We found that
salmonid IL-6Rα had a large D1 domain mainly due to an insertion of two amino acid repeat sequences. We
discovered that the number of each of the two repeats was variable in four trout cell lines studied and in
different fish. Finally, we examined the expression of IL-6Rα and gp130 in tissues from healthy fish and their
modulation by LPS and Poly I:C in the cell lines.
Although the fish IL-6Rα and gp130 molecules were quite divergent (Tables 2 and 3), they showed similar
domain structure, i.e. 3 and 6 domains, respectively, in the extracellular regions, with a WSXWS motif at the
C-terminal (D3) of the cytokine binding domain (CBD), as seen in mammals. Key intracellular motifs were
also conserved in gp130 and included the box-1-3, critical for Jak association and signalling, the docking sites
for other downstream molecules, such as the YXXQ motif for STAT3 and the YSTV motif for SHP-2 (Fig. 2).
Phylogenetic tree analysis (Fig. 3) grouped the fish molecules with their tetrapod counterparts with high
bootstrap support, adding further confirmation that the molecules found are the orthologues to mammalian IL6Rα and gp130. However, important differences were observed, including a large D1 domain in fish IL-6Rα,
and a different potential disulphide bond pattern in both IL-6Rα and gp130, that will be discussed below.
Domain structure prediction and multiple alignment (Fig. 1) suggested the Ig-like D1 domain in all fish IL6Rα molecules is bigger than in tetrapods. The salmonid IL-6Rα sequences were further enlarged in this
domain by the insertion of two different repeat motifs corresponding to the 13 aa peptide sequences [TS][TF]-VSTTT-[ND]-TTSNG and TTVS-[AT]-IKD-[DG]-S-[KD]-N-[GR] respectively. The numbers of these
repeats were different in different cell lines and individual fish (Fig. 4), suggesting that salmonid IL-6Rα has a
highly polymorphic D1 domain with the potential to affect responsiveness to IL-6. The Ig-like D1 domain of
mammalian IL-6Rα is important for intracellular transport through the secretory pathway and is also involved
in the noninduced shedding of the receptor to release a soluble IL-6Rα. However, deletion of the D1 domain
of human IL-6Rα does not affect its bioactivity, suggesting that the D1 domain is not critical for IL-6 binding
(Vollmer el al. 1999). Thus a large D1 domain in the fish IL-6Rα may be tolerable for its activity. However,
the large D1 domain in fish, and particularly in salmonids, may have as yet unknown functional impacts and
for example, could affect receptor secretion, membrane expression and shedding, or the stability of the IL6/IL-6R signalling complex.
Structural studies have revealed that IL-6 is first engaged by IL-6Rα and then presented to gp130 in the proper
geometry to facilitate a cooperative transition into the high-affinity, signalling-competent hexamer. Human
IL-6 possesses a total of five points of interaction with its receptor. The site I of the protein is able to contact
several residues located between the D2 and D3 domains in the IL-6Rα chain, with the D3 domain providing
the majority of the contact surface. Sites IIa and IIb of IL-6 interact with amino acids present between gp130
14
domains D2 and D3, and between the D3 domains of gp130 and IL-6Rα, respectively. The IL-6 site IIIa links
to the gp130 D1 domain, and the site IIIb interacts with the gp130 D1 domain and the IL-6Rα D2 domain
(Boulanger et al. 2003). Thus the D2-3 of IL-6Rα and D1-3 of gp130 are critical to form a signalling complex
with mammalian IL-6. The well conserved D2-3 of IL-6Rα (Fig. 1) and D1-3 of gp130 (Fig. 2) across
vertebrates suggests that a similar signalling complex to that seen for mammalian IL-6/IL-6R may be
conserved in fish.
Soluble IL-6Rα can be produced by shedding of membrane bound receptor or by translation from alternatively
spliced transcripts, as seen in humans where two transcripts occur as a result of alternative splicing, that
translate into a membrane-bound form and its soluble counterpart (Oh et al. 1996). Alternative splicing
variants that produce soluble IL-6Rα, or soluble IL-11Rα have not been found in chicken, as shown by RTPCR/Southern blot analysis where no transcripts shorter than the full-length chicken IL-6Rα are seen
(Nishimichi et al. 2006; Kawashima et al. 2005). In trout a single band was amplified using primers located in
the 5’ and 3’-UTR of the IL-6Rα sequence during our cloning procedure, indicating that alternative splicing to
produce a soluble IL-6Rα is unlikely to occur in trout either.
Intra-chain disulphide bonds between the thiol groups of two peptidyl-cysteine residues can contribute to
protein folding and stability of cytokines and their receptors. Thus the N-terminal FNIII domain of the CBD in
cytokine receptors, at least in mammals, contains two pairs of conserved cysteines arranged in a CX-(9-10)CXWX-(26-32)-CX-(10-15)-C motif linked by two disulphide bonds (Liong and Ward 2007). The tetrapod IL-6Rα
have four conserved disulphide bonds, one in the D1 domain, two in the D2 domain and one that connects the
D1 and D2 domains (Cole et al. 1999; Varghese et al. 2002). All the cysteine residues for the four disulphide
bonds are conserved in the IL-6Rα molecule across vertebrates (Fig. 1). In addition, all the fish IL-6Rα
molecules possess a fifth pair of cysteine residues in the D2 domain that may form an additional disulphide
bond to stabilise their tertiary structure. Human gp130 contains two pairs of disulphide bonds in each of the
D1 and D2 domains (Moritz et al., 2001), and the cysteine residues involved are conserved in gp130
molecules across tetrapods. In contrast to the fish IL-6Rαs that possess an extra cysteine pair, fish gp130
molecules lack two pairs of the cysteine residues conserved in tetrapods, one in each of the D1 and D2
domains.
N-glycosylation is the most ubiquitous protein co-translational modification in the endoplasmic reticulum.
This modification serves as a primary determinant for specific molecular recognition as well as protein folding
and stability, and therefore is an essential and highly conserved protein modification pathway in eukaryotic
cells. Multiple potential N-glycosylation sites have been predicted in both trout IL-6Rα and gp130
(Supplementary Figs. 1–2). Although we have not observed the conservation of N-glycosylation sites across
vertebrates in the multiple alignment of IL-6Rα, two conserved sites, one each in the D1 and D2 domains,
were identified in gp130 across vertebrates (Fig. 2). Human gp130 is N-glycosylated at 9 of 11 potential sites.
15
Mutation analysis revealed that mutein gp130 (with N-glycosylation sites removed) is not efficiently
transported to the plasma membrane but is degraded in the proteasome. However, the small quantities of
mutein gp130 which do reach the cell surface, are still able to transduce the IL-6 signal (Waetzig et al. 2010).
Thus, N-linked glycosylation of gp130 is required for molecular stability but not the signal-transducing
function, and the conservation of potential N-glycosylation sites across vertebrate gp130 molecules concurs
with this. Interestingly, the salmonid IL-6Rα molecules possess polymorphic numbers of two 13 aa repeat
sequences and each repeat motif contains a potential N-glycosylation site. Thus salmonid IL-6Rα from
different individuals has different N-glycosylation potential as the number of repeats is variable.
In mammals, gp130 is expressed ubiquitously, whilst the IL-6Rα chain is expressed only by hepatocytes,
phagocytes and some lymphocytes (Saito et al. 1992). In trout a ubiquitous expression of gp130 was detected
in all the 14 tissues examined (Fig. 5). Interestingly, the expression of trout IL-6Rα was comparable to or even
higher than that of gp130 expression in some tissues. In contrast, the IL-6Rα expression was lower than gp130
in most of the cell lines examined (epithelial RTL and fibroid RTG-2 and RTGill), except the
monocyte/macrophage like cell line (RTS-11) where comparable expression of both IL-6R subunits was seen.
The relatively high level expression of IL-6Rα in tissues may be due to its expression in leucocytes, since the
expression of IL-6Rα was low in the epithelial and fibroid cell lines (Fig. 6). The bioactivities of recombinant
trout IL-6 have only been detected in RTS-11 cells (Costa et al. 2011) that express a high level of IL-6Rα and
not in the other cell lines. Trout IL-6 is also active on leucocytes prepared from head kidney and spleen that
similarly express high levels of IL-6Rα (unpublished data). Such data suggest that high level transcript
expression of IL-6Rα may be critical for responsiveness to IL-6.
The trout IL-6Rα was highly expressed in immune tissues such as the spleen and head kidney, and was also
expressed in liver, gills, brain, heart, thymus and intestine. This is in line with the pleiotropic functions of IL-6
in the regulation of the immune response, inflammation, and hematopoiesis, as well as in the (liver) acute
phase response. IL-6 was originally described as a T-cell-derived B-cell differentiation factor, essential for
antibody production by activated B cells (Nishimoto and Kishimoto 2006). It also affects other immune cells,
and induces T-cell activation and differentiation and macrophage differentiation. IL-6 is one of the
differentiating factors for Th17 cells (Betteli et al. 2007), and switches the differentiation of monocytes from
dendritic cells to macrophages (Chomarat et al. 2000). Moreover, the role of IL-6 in the production of acute
phase proteins in liver, as well as its role in hepatic regeneration, has been reported (Bayne and Gerwick 2001;
Drucker et al. 2010). Therefore, lymphocytes, monocytes/macrophages and hepatocytes all require the
presence of the IL-6Rα chain to bind and respond to this cytokine.
The highest expression level of trout gp130 was found in the muscle (Fig. 5). Mammalian gp130 is highly
expressed in the sarcolemma and also in fibroblasts (Ancey et al. 2003), and is involved in myocyte
hypertrophy and fibroblast proliferation (Fredj et al. 2005), suggesting an important role of IL-6 family
cytokines in muscle. This result is in contrast to the expression of a related gene (JfGPH) in Japanese flounder
16
(Santos et al., 2007), thought to be an ancestral type I cytokine receptor found in teleost fish, where highest
expression of JfGPH was found in immune tissues by RT-PCR. Trout gp130 was only expressed at a low
level in intestine, gills, thymus and kidney, as revealed by real-time RT-PCR. This may represent a real
difference between the molecules or the fish species, as also seen with the salmonid IL-6Rα chain that has an
extra-large D1 domain and that is highly expressed, at least at the transcript level. Thus, in salmonids perhaps
a controlled expression of gp130 helps to regulate IL-6 function.
The secretion of mammalian IL-6 is induced by inflammatory stimulants such as LPS (Ghezzi et al. 2000),
and IL-6 can up-regulate its own receptor expression (Thabard et al. 2001). In trout, IL-6 expression in RTS11 cells, as well as in primary macrophages is induced by LPS, Poly I:C and the proinflammatory cytokine IL1β (Costa et al. 2011). With the aim to analyse if similar mechanisms could regulate IL-6R expression in
rainbow trout, we have analysed the expression of IL-6Rα and gp130 after stimulation. No change in
expression level was observed after trout rIL-6 stimulation of RTS-11 cells (data not shown), suggesting that
different mechanisms may be involved in the modulation of IL-6 receptor expression in fish. Despite being
refractory to IL-6, the expression of both IL-6Rα and gp130 was modulated by LPS and Poly I:C in a cell line
and time dependent manner. A modest increase of IL-6Rα expression was seen in RTS-11, RTL and RTG-2
cells after LPS stimulation, and a modest increase of gp130 expression was seen in RTG-2 and RTGill cells
after Poly I:C stimulation (Fig. 6). However, gp130 expression was down-regulated (at 8 h) by both LPS and
Poly I:C in RTS-11 cells, that express high levels of both IL-6Rα and gp130, and are known to be responsive
to trout IL-6. The down-regulation of gp130 in trout macrophage may represent a control mechanism to limit
their responsiveness to IL-6, perhaps in terms of autocrine signalling. It is interesting to note that Poly I:C also
down-regulates the expression of JfGPH in leukocytes prepared from spleen, kidney and peripheral blood as
well as in a natural embryo (HINAE) cell line (Santos et al. 2007). Clearly more work needs to be done on
different cell populations and in parallel situations in different species to fully establish the mechanisms by
which IL-6 receptor expression can be modulated in fish.
Acknowledgments
The work was supported by a European Community project IMAQUANIM (FOOD-CT-2005-007103).
M.M.C. thanks the Consejo Superior de Investigaciones Científicas (CSIC, Spain) and the Xunta de Galicia
for her “Ángeles Alvariño” postdoctoral contract. M.M.M. thanks the FCT (Foundation for Science and
Technology, Portugal) and POPH/FSE for her PhD studentship (Grant no. SFRH/ BD/ 38236/ 2007).
17
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Figure legends.
Figure 1. Multiple alignment of the deduced amino acid sequences of the trout IL-6Rα chain with
selected IL-6Rα molecules from other species. The multiple alignment was produced using ClustalW2 and
conserved amino acids shaded using BOXSHADE (V3.21). The signal peptide (SP), and the transmembrane
domain (TM) are indicated by solid lines above the alignment. The highlighted features include the ten
conserved cysteine residues in D1 and D2 domains, the two proline rich hinge regions (boxed) that separate
the three domains (D1-3), and the WSXWS motif in the C-terminal of D3 domain in the extracellular region.
The two sets of 13 amino acids located in the Ig-like D1 domain of salmonid IL-6Rα were indicated by a
black box.
Figure 2. Multiple alignment of the deduced amino acid sequences of trout gp130 with selected gp130
molecules from other species. The multiple alignment was produced using ClustalW2 and conserved amino
acids shaded using BOXSHADE (V3.21). The signal peptide (SP), and the transmembrane domain (TM) are
indicated by solid lines above the alignment. The six modular domains (D1 to 6) separated by the five prolinerich hinge regions are indicated. The features in the extracellular region include the eight conserved cysteine
residues, the two conserved N-glycosylation sites (black bar) in the D1 and D2 domains, and the WSXWS
motif in the D3 domain. The conserved three boxes (box-1 to 3), the serine rich region, the di-leucine
dependent receptor internalisation motif, and the YSTV and four YXXQ (YXXQ1-4) motifs in the
intracellular region are also highlighted.
Figure 3. Phylogenetic tree analysis of the IL-6Rα (A) and gp130 (B) chains and their relatives. The
evolutionary history was inferred using the Maximum Likelihood method based on the JTT matrix-based
model using the MEGA5 program. The tree with the highest log likelihood is shown. The tree is drawn to
scale, with branch lengths measured in the number of substitutions per site. Node values represent percent
bootstrap confidence derived from 2,000 replicates. The molecule and the common species name are followed
by the accession number. The new sequences reported in this review are shaded.
Figure 4. PCR (A and B) and sequence (C) analysis of the two amino acid repeat sequences in the Iglike D1 domain of rainbow trout IL-6Rα. cDNA samples were prepared from different tissues (A) and cell
lines (B) and subjected to PCR amplification as described in the Materials and Methods. The resultant
products were revealed on 1% agarose gels containing ethidium bromide. The results from two fish and two
samples from each cell line are shown. Note the two products seen with Fish B and the different sizes seen in
different cell lines. Selected PCR products were cloned and sequence analysed (C). The two repeat sequences
were found to have different frequencies in different fish and cell lines, with the first 39 bp repeat encoding
for the 13 aa consensus [T/S]-[T/F]-VSTTT-[D]-TTSNG, and the second 39 bp repeat encoding for the 13 aa
consensus TTVS-[A/T]-IKD-[D/G]-S-[K/D]-N-[G/R].
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Figure 5. The expression of IL-6Rα and gp130 in vivo. The expression of IL-6Rα and gp130 was analysed
in 14 tissues from healthy fish by real-time PCR. The expression of transcripts was normalized against that of
the housekeeping gene EF-1α and expressed as arbitrary units (A), where 1 arbitrary unit was equal to the
average expression level of gp130 in intestine, which had the lowest expression level among the tissues
studied. The ratio of the expression level of IL-6Rα and gp130 in the same tissue is presented in (B), where
the number over the bar is the average expression level of IL-6Rα divided by that of gp130. Data represent the
average + SEM from six healthy fish. Significant results of a paired sample t-test between the expression
levels of IL-6Rα and gp130 in the same tissue is shown above the bars as: *p<0.05, **p≤0. 01 and
***p≤0.001.
Figure 6. Modulation of IL-6Rα and gp130 expression in four trout cell lines. The relative constitutive
expression of IL-6Rα and gp130 in four trout cell lines RTL, RTG-2, RTGill and RTS-11, was examined by
real-time PCR and expressed as arbitrary units (A), where 1 arbitrary unit was equal to the average expression
of IL-6Rα in the RTL cell line, which had the lowest expression level among the cell lines tested. The four
trout cell lines were then stimulated with lipopolysaccharide (LPS, 25 µg/ml) and polyinosinic: polycytidylic
acid (Poly I:C, 50 µg/ml) for 4, 8 and 24 h and the expression of IL-6Rα (B) and gp130 (C) also examined by
real-time RT-PCR and expressed as a fold change relative to time-matched controls. The p-value of a LSD
post hoc test after a significant one way-analysis of variance between the stimulated and time matched
controls is shown above the bars as: *p < 0.05; **p≤0.01; ***p≤0.001.
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