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Expression profiling and functional characterization of galectin-3 of turbot (Scophthalmus maximus L.) in host mucosal immunity
Accepted Manuscript Expression profiling and functional characterization of galectin-3 of turbot (Scophthalmus maximus L.) in host mucosal immunity Mengyu Tian, Ning Yang, Lu Zhang, Qiang Fu, Fenghua Tan, Chao Li PII:
S1050-4648(18)30633-8
DOI:
10.1016/j.fsi.2018.10.009
Reference:
YFSIM 5610
To appear in:
Fish and Shellfish Immunology
Received Date: 4 August 2018 Revised Date:
12 September 2018
Accepted Date: 5 October 2018
Please cite this article as: Tian M, Yang N, Zhang L, Fu Q, Tan F, Li C, Expression profiling and functional characterization of galectin-3 of turbot (Scophthalmus maximus L.) in host mucosal immunity, Fish and Shellfish Immunology (2018), doi: https://doi.org/10.1016/j.fsi.2018.10.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Expression profiling and functional characterization of
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galectin-3 of turbot (Scophthalmus maximus L.) in host
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mucosal immunity
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Mengyu Tiana,1, Ning Yang a,1, Lu Zhanga, Qiang Fua, Fenghua Tana, Chao Lia,*,
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266109, People’s Republic of China
Marine Science and Engineering College, Qingdao Agricultural University, Qingdao
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All these authors contributed equally to this work.
Corresponding author: Chao Li, [email protected]
ACCEPTED MANUSCRIPT Abstract: Galectins, a family of evolutionary conserved β-galactoside-binding
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proteins, have been characterized in wide range of species. Galectin-3 is the only
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member in the chimera type, which is monomeric lectin with one CRD domain. A
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growing body of evidence have indicated vital roles of galectin-3 in innate immune
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responses against infection. Here, one galectin-3 gene was captured in turbot
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(SmLgals3) contains a 1,203 bp open reading frame (ORF). In comparison to other
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species, SmLgals3 showed the highest similarity and identity to large yellow croaker
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and medaka, respectively. The genomic structure analysis showed that SmLgals3 had
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5 exons similar to other vertebrate species. The syntenic analysis revealed that
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galectin-3 had the same neighboring genes across all the selected species, which
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suggested the synteny encompassing galectin-3 region during vertebrate evolution.
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Subsequently, SmLgals3 was widely expressed in all the examined tissues, with the
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highest expression level in brain and the lowest expression level in skin. In addition,
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SmLgals3 was significantly down-regulated in intestine following both Gram-negative
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bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae
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immersion challenge. Finally, the rSmLgals3 showed strong binding ability to all the
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examined microbial ligands. Taken together, our results suggested SmLgals3 plays
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vital roles in fish innate immune responses against infection. However, the knowledge
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of SmLgals3 are still limited in teleost species, further studies should be carried out to
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better characterize its detailed roles in teleost mucosal immunity.
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Keyword: Galectin-3; turbot; Vibrio anguillarum; Streptococcus iniae; microbial
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ligand binding
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1. Introduction
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Galectins, a family of evolutionary conserved β-galactoside-binding proteins, have been characterized in a wide range of species, including fungi, parazoa, and
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vertebrates [1, 2]. The galectins were featured with a characteristic carbohydrate
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recognition domain (CRD) with an affinity for β-galactosides, and a conserved
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sequence motif [3]. According to the structural features, galectin family has been
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classified into three different types: proto-type, Chimera type and tandem-repeat type
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[4]. As a group of carbohydrate-binding proteins, galectins could involve in many
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biological processes, such as immune responses, inflammation, cell migration and
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development [5]. So far, fourteen galectin members have been discovered in
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mammals, but lower vertebrates and invertebrates appear to have a smaller number of
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galectins. In teleost, several studies have been carried out to characterize galectin
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genes, since the first galectin gene was identified in electric organs of the electric eel
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(Electrophorus electricus) [6]. For instance, eight galectins were identified in catfish,
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and showed significant expression changes following Edwardsiella ictaluri and
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Flavobacterium columnare challenge in mucosal tissues [7].
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Among three galectin types, only galectin-3 belongs to the chimera type, which are
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monomeric lectins with one CRD domain. Similar to the other galectin types,
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galectin-3 also lacks a signal sequence which is required for secretion, but the
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galectin-3 protein could be released into the extracellular space [8]. Of note,
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galectin-3 has been captured in a wide range of immune cells, including mast cells,
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macrophages, neutrophils, monocytes and T cells. For instance, galectin-3 deletion
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suppressed production of pro-inflammatory cytokines and significantly reduced
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activation of NOD-like receptor family in macrophages in mice [9]. In T cells,
ACCEPTED MANUSCRIPT galectin-3 could induce the production of IL-2 [10], as well the apoptosis in both Th1
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and Th2 cells [11]. In teleost species, galectin-3 could reduce the adhesion of
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infectious hematopoietic necrosis virus by direct interaction with virus envelop on the
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epithelial cell surface in a carbohydrate-dependent manner [12]. In oyster
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(Crassostrea gigas), galectin-3 displayed agglutination ability against both
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Micrococcus luteus and Pichia pastoris [13]. However, most of the studies of
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galectin-3 were performed in mammals, the characterization of galectin-3 in teleost
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was still limited in a handful species.
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Turbot (Scophthalmus maximus L.), one of the most extensively maricultured
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species in China, suffers from the bacterial disease including Vibrio anguillarum and
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Streptococcus iniae. Many studies have been performed to identify the
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immune-related genes and investigate their associated activities during bacterial
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infection [14-18], for further development of the disease control and prevention
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measures in turbot. In this regard, we sought here to characterize galectin-3 gene in
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turbot, and its expression patterns following different bacterial infection, as well as
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microbial ligand-binding activities to gain initial insight into the immune roles of
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galectin-3 in turbot.
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2. Materials and methods
2.1. Sequence identification and analysis
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The turbot galectin-3 gene was captured from transcriptome database [19] by
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BLAST program using galectin-3 protein sequences from other species as queries.
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The retrieved candidate sequences were then translated using ORF Finder
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further verified against NCBI non-redundant protein sequence database by BLASTP
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(http://blast.ncbi.nlm.nih.gov/Blast.cgi). The conserved domains and signal peptides
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were further identified by the simple modular architecture research tool (SMART;
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http://smart.embl-heidelberg.de/). The theoretical pI, molecular mass and
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N-glycosylation sites were captured in ExPASy server [20]. The intron and exon
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structures were predicted by Splign program [21].
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2.2. Phylogenetic analysis
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The phylogenetic tree was constructed based on the amino acid sequences of galectin-3 gene from various species including human (Homo sapiens), shrew mouse
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(Mus pahari), chicken (Gallus gallus), green sea turtle (Chelonia mydas), spotted gar
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(Lepisosteus oculatus), zebrafish (Danio rerio), medaka (Oryzias latipes), Tilapia
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(Oreochromis niloticus), fugu (Takifugu rubripes). The multiple protein sequence
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alignment was performed in Clustal Omega program [22]. A neighbor-joining
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phylogenetic tree was generated using the Molecular Evolutionary Genetics Analysis
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(MEGA 6) [23].
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2.3. Syntenic analysis
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Syntenic analysis was performed to further verify the identification of turbot
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galectin-3 gene. Briefly, the protein sequences of neighboring genes of the turbot
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galecin-3 were predicted from the turbot scaffold using FGENESH program. The
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identified protein sequences were annotated by BLASTP program against NCBI
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non-redundant (nr) database. The conserved syntenic pattern of galectin-3 gene in
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other species were characterized in Ensembl database and Genomicus [24].
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2.4. Bacteria challenge and sample collection
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In order to characterize the roles of galectin-3 gene in the host immune responses against bacterial infection, the Gram-negative bacteria V. anguillarum and
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Gram-positive bacteria S. iniae were selected to conduct the immersion challenge.
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Turbot fingerlings (average body weight: 15.6 g and average body length = 5.5 cm)
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were obtained from the turbot hatchery (Haiyang, Shandong, China), and acclimated
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in the laboratory in a flow-through system for at least two weeks prior to challenge.
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After a pre-challenge, the bacteria was re-isolated from single symptomatic fish and
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biochemically confirmed before experiment. During the experiment, the experimental
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fish were challenged for 2 h by immersion, and then transferred in fresh water. At
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each timepoint following challenge, skin, gill and intestine samples were collected
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from 15 fish (5 fish per pool) from the appropriate aquaria.
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Briefly, the V. anguillarum was inoculated in LB broth in a shaker (180 rpm) at
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28 °C overnight. The fish were immersed at a final concentration of 5×107 CFU/mL
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for 2 h, while the control fish were immersed in sterilized media alone. Aquaria were
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randomly assigned for 2 h, 6 h, 12 h and 24 h post-treatment and 0 h control with
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thirty fish in each aquarium for sample collection.
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The S. iniae isolate was inoculated in LB medium in a shaker incubator at 28 °C
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overnight. The fish were equally divided into five aquariums, 4 challenged groups (2
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h, 4 h, 8 h and 12 h) and one control group. For the challenge, the fish were immersed
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for 2 h at a final concentration of 5×106 CFU/mL, while the control fish were
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immersed in sterilized media alone. All samples from both experiments were
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flash-frozen in liquid nitrogen and then stored in a -80 °C ultra-low freezer until
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preparation of RNA.
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2.5. Total RNA extraction and cDNA synthesis
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Prior to RNA extraction, tissue samples were homogenized under liquid nitrogen
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using mortar and pestle. Total RNA was extracted using Trizol® Reagent (Invitrogen,
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USA) according to the supplied protocol. The quality and quantity of RNA of each
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sample were measured on a Nanodrop 2000 (Thermo Electron North America LLC,
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FL). All extracted samples had an A260/280 ratio greater than 1.8, and were diluted to
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250 ng/µl.
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2.6. Real-time PCR analysis
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Gene specific primers were designed using Primer3 software based on the turbot galectin-3 sequences, and 18S rRNA gene was used as a reference gene (Table 1).
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First strand cDNA was synthesized by PrimeScript RT reagent Kit (TaKaRa)
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according to manufacturer’s protocol (500 ng RNA per 10 µl reaction). Quantitative
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real-time PCR (qPCR) was performed on a CFX96 real-time PCR detection system
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(Bio-Rad Laboratories, Hercules, CA) using the SYBR ExScript qRT-PCR Kit
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(Takara, Dalian, China). The reaction systems for all real-time PCR were as follows:
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1.0 µL of each primer (5 µM), 5.0 µL SYBR Green supermix, 2.0 µL
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RNase/DNase-free water, and 1.0 µL 200 ng/µL cDNA. The PCR reaction mixture
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was denatured at 95 °C for 30 s and then subjected to 40 cycles of 95 °C for 5 s, 58 °C
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ACCEPTED MANUSCRIPT for 5 s and followed by dissociation curve analysis, 5 s at 65 °C, then up to 95 °C at a
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rate of 0.1 °C/s increment, to verify the specificity of the amplicons. Results were
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analyzed using Relative Expression Software Tool (REST) to capture the significance
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at the level of P<0.05 [25]. In order to determine the gene expression patterns in
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turbot healthy tissues, the tissue with the lowest Ct values were used as control. The
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mRNA expression levels of all samples were normalized to the levels of 18S
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ribosomal RNA gene in the same samples. A no-template control was run on all
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plates.
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2.7. Plasmid construction
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In order to construct the expression plasmid for SmLgals3, SmLgals3 was amplified with the specific primers following cDNA synthetization. After gel extraction, the
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PCR products were ligated to pEASY-Blunt-E1 vector, and then transformed into
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competent Trans1-T1 cells. Following blue-white spotting selection, the positive
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clones were selected and sequencing with T7 Promoter Primer. The verified
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recombinant plasmid was extracted and marked as pEASY-E1- Lgals3.
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2.8. Expression and purification of recombinant SmLgals3
The recombinant plasmid pEASY-E1- Lgals3 was transformed into E.coli BL21
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(DE3). The transformant BL21- Lgals3 and the control BL21 with empty pET-32a
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were cultured in LB medium, and induced by adding 0.5 mM
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isopropyl-b-D-thiogalactopyranoside. The expressed protein was purified by
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nickel-nitrilotriacetic acid chromatography, and analyzed by 12% sodium dodecyl
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sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by staining
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with Coomassie brilliant blue. The concentration of the recombinant protein was
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determined using Bradford's method.
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2.9. Solid-phase enzyme-linked immunosorbent assay (ELISA)
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The binding ability of rSmLgals3 with lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN) was detected by ELISA method. Briefly, LPS/LTA
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/PGN (5 µg/ml) were coated to 96 microtitor plate at 4 °C overnight. The wells were
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washed with 300 µl PBST three times, and then blocked with 100 µl 5% BSA at 4 °C
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for 1 h. Then, 100 µl of increasing concentrations of purified recombinant SmLgals3
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(0.5, 1, 2, 4, 8 and 16 µg/mL) were added into each ligand-coated well, with 4
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replicates for each concentration, and incubated at 37 °C for 1h. Subsequently, the
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wells were incubated at 37 °C for 1 h with 100 µl mouse anti-His antibody (Solarbio,
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Beijing, China) (diluted 1:1000 in 5% BSA), followed by another incubation at 37 °C
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for 40 min with the addition of 100 µl horseradish peroxidase conjugated goat
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anti-mouse IgG (Solarbio, Beijing, China) (diluted 1:1000 in 5% BSA). Finally, the
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reaction was terminated by adding 0.5M sulfate, and the plate was then read at 450
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nm with an ELISA reader.
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3. Results
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3.1 Identification of turbot galectin-3 genes
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After searching the turbot transcriptome and genome database [26] using available
ACCEPTED MANUSCRIPT galectin-3 sequences from other species as queries, one galectin-3 gene was captured
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in turbot (SmLgals3). In detail, the full-length SmLgals3 (GenBank accession:
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MF797865) transcript contains a 1,203 bp open reading frame (ORF) encoding 400
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amino acids residues with a predicted molecular mass of a 39.74 kDa and a theoretical
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isoelectric point of 5.12 (Table 2). The deduced SmLgals3 protein was predicted to
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have 1 Casein kinase II phosphorylation sites, as well as 24 negatively charged
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residues (Asp + Glu), 13 positively charged residues (Arg + Lys), and with an
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instability index of 54.9 and aliphatic index of 45.42 (Table 2). In comparison to other
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species, SmLgals3 showed the highest similarity to large yellow croaker (68%),
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followed by zebrafish (66.5%), while the highest identity was observed with medaka
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(61.3%), followed by tilapia (60.1%) (Table 3).
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3.2. Genomic structure analysis of SmLgals3 gene
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Following sequence identification, the genomic architecture of SmLgals3 was also investigated. Following the comparison of the exon/intron organization of
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SmLgals3 with other vertebrates, the N-terminal and C-terminal showed different
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similarities among the selected species. In detail, the exon number was varied from 5
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to 7, and the CRD domain was composed by three same exons (89 bp,166 bp and 156
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bp) in all the species (Fig.1), whereas the different number of exons were observed in
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their N-terminal domains. In general, there were 5 exons in the mammals and chicken,
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and more exons (6 and 7) in fish species, except turbot also showed 5 exons (Fig.1).
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3.3. Phylogenetic analyses
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Subsequently, the phylogenetic analysis was performed with amino acid sequences of galectin-3 proteins from species of fish and mammals, using the
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neighbor-joining method in MEGA 6. In our results, SmLgals3 was firstly clustered
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with fugu, and then clustered with tilapia, medeka, and zebrafish (Fig. 2). The higher
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vertebrates formed single clade including human, mouse, chicken and green sea turtle
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(Fig. 2). And all branching nodes were supported by high bootstrap values.
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3.4. Syntenic analysis
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Finally, the syntenic analysis was performed for further validation of the
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identification of SmLgals3 gene. In general, a relatively conserved synteny was
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observed in comparison of SmLgals3 to other species. After searching the genomic
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databases of different species, the same neighboring genes were found across the
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selected species, including CDKN3, CNIH1, GMFB, L2HGDH, MED6, SOS2, and
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ACTN1 (Fig. 3).
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3.5. The tissue distribution of SmLgals3
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The tissue distribution of SmLgals3 was examined in eight turbot healthy tissues by
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real-time PCR method. In our results, SmLgals3 was widely expressed in all the
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examined tissues, with the highest expression level in brain (46.28 fold), followed by
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intestine (12.6 fold), head-kidney (7.15 fold) and blood (6.2 fold), while the lowest
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expression level was detected in skin (Fig. 4).
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3.6. Expression profiles of SmLgals3 following bacterial challenge
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In order to characterize the immune roles of SmLgals3 in turbot mucosal immunity, the expression profiles of SmLgals3 were investigated in mucosal tissues (gill, skin
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and intestine) at early timepoints following immersion challenge with Gram-negative
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bacteria V. anguillarum, and Gram-positive bacteria S. iniae, respectively.
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Following V. anguillarum challenge, SmLgals3 was significantly down-regulated in all the three tissues except up-regulated in skin with 12.36 fold at 2 h, but it was
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quickly down-regulated with -4.65 fold at 6 h, and then returned to basal level (Fig. 5).
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The most up-regulation was detected in intestine at 6 h with -28.19 fold, and -6.59
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fold at 12 h. Different from other tissues, the gill showed -3.58 fold at 6 h, -9.64 fold
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at 12 h, and the most dramatic down-regulation at 24 h with -22.99 fold (Fig. 5).
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In S. iniae challenge, the SmLgals3 was down-regulated much more quickly than that in V. anguillarum challenge. The SmLgals3 was down-regulated at 2h at all the
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three tissues, with -13.01 fold in intestine, -10.78 fold in skin, and -3.43 fold in gill
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(Fig. 6). Interestingly, it quickly returned to basal level in gill and skin, while continue
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to be down-regulated in intestine with -9.58 fold at 8h and -15.46 fold at 12h (Fig. 6).
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3.7. Microbial ligand-binding in vitro
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The binding ability of SmLgals3 to microbial ligands was also investigated to
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further characterize its function. Briefly, the rSmLgals3 was purified from E. coli as a
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native His-tagged protein. In SDS-PAGE analysis, only a single band was observed
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(Supplementary Fig. 1). In our results, the strong binding ability was observed to LPS,
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PGN and LTA (Fig. 7). The strongest binding ability was detected to LPS which
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showed significant binding ability at 0.5µg/ml, while the significant binding ability
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for PGN was observed at 0.5µg/ml, and 2.0 µg/ml for LTA (Fig. 7).
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4. Discussion
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Galectins are widely distributed in primary and secondary lymphoid organs and
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many types of immune cells, with vital roles in regulation of immune cell
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proliferation and apoptosis [27]. A growing body of evidence have indicated
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galectin-3 could involve in chemotaxis, phagocytosis, LPS-induced IL-1 production,
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NADPH-oxidase activity, and IL-4 induced survival of memory B cells [28-30].
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Although considerable experimental evidences have elucidated galectin functions
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within the immune system in higher mammals, the characterization of galectin-3 is
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still lacking in teleost species. Here, we identified galectin-3 gene in turbot,
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performed expression profiling analysis of galectin-3 in response to different bacterial
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infection, as well as the binding ability analysis to different microbial ligands, to
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provide initial information for further functional characterization of galectin-3 in
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teleost. In this study, one galectin-3 gene was captured in turbot (SmLgals3) with
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similar molecular properties to other fish species (Table 1). As an evolutionarily
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highly conserved family of proteins, the genomic structure analysis, phylogenetic
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analysis and synteny analysis validated the identification of SmLgals3 and showed the
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strong orthology to their counterparts vertebrate species.
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In tissue distribution analysis, SmLgals3 was widely expressed in all the examined
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tissues, with the highest expression level in brain, followed by intestine (Fig. 4).
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Interestingly, galectin-3 was reported to be widely expressed in rat brain, and seems to
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be regulated by developmental cascades [31], also was considered as mediator of
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microglia responses in injured brain [32]. In addition, galectin-3 was highly expressed
ACCEPTED MANUSCRIPT in the colons of patients with ulcerative colitis [33]. However, galectin-3 was highly
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expressed in gill and skin, and lowly expressed in intestine [34], while it showed the
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lowest . With the limited information about expression profiling of galectin-3 in
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teleost, the putative roles of galectin-3 in different tissues of teleost need to be further
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characterized.
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Subsequently, the expression patterns of SmLgals3 were characterized following
different bacteria challenge in order to gain insight into its immune roles in mucosal
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immunity. Notably, SmLgals3 showed significant down-regulation in all the tissues
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following both Gram-negative bacteria V. anguillarum, and Gram-positive bacteria S.
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iniae challenge, with the most down-regulation in intestine. Previously, V.
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anguillarum cells were detected in spleens from more than 50% of orally or rectally
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infected fish, suggesting that the intestine might serving as a portal of entry for V
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anguillarum [35]. Following orally administered V. anguillarum in turbot, the bacteria
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showed strong ability to survive in the acidic environment of the stomach, and persist
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in the intestine, which suggested that the turbot intestinal tract could serve as an
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enrichment site for V. anguillarum [36]. In zebrafish, the entry of V. anguillarum
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through the mouth into the intestine was detected at 2h following bath challenge with
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V. anguillarum, and the localisation of V. anguillaum was detected in intestine at 6h
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[37]. Moreover, following bath-vaccination of live attenuated V. anguillarum vaccine,
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bacteria cells proliferated rapidly in 3h after vaccination and maintained at a high
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level until 6h in the intestine, with a significant up-regulation of TLR5 triggering
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MyD88-dependent signaling pathway in the intestine [38]. Comparing to V.
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anguillarum challenge, SmLgals3 was significantly down-regulated in all the
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timepoints in the intestine following S. iniae challenge. In Japanese flounder, the S.
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iniae bath challenge could induce much more significant deaths in lower
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ACCEPTED MANUSCRIPT concentration than oral challenge [39]. Previously, several immune-related genes were
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also significantly up-regulated following S. iniae infection in turbot intestine [14-16].
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In contrast, galectin-3 was significantly up-regulated in catfish skin following
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Aeromonas hydrophila infection [40]. Although our knowledge of galectin-3 functions
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are limited in fish, it has been well documented in mammalian species. For instance,
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In gastric epithelial cells, galectin-3 was up-regulated following Helicobacter pylori
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infection [41]. Ggalectin-3 showed the inhibition effect on the colonic mucosa
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inflammation and reduced disease severity by inducing regulatory T cells [42].
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Galectin-3 deletion resulted in the suppressed production of pro-inflammatory
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cytokines in colonic macrophages and favored their alternative activation, as well as
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significantly reducing activation of NOD-like receptor family in macrophages [33].
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Several studies following different bacteria challenge, suggested that galectin-3 may
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act as a cell surface docking site or a cross-linking molecule promoting adhesion
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[43-46]. Further work is warranted to examine whether turbot galectin-3 may play
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similar roles in supporting pathogen adhesion.
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As typical lectins, galectins possessed carbohydrate binding ability to microbial
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pathogens by recognizing exogenous ligands, especially carbohydrates on the surface
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of microbes via their CRDs, and subsequently trigger downstream immune signaling
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pathways to eliminate the pathogens. The structure features might affect their
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specificity and binding affinity to carbohydrates. As the only member of chimera type
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galectins, galectin-3 should have different immune functions compared to proto and
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tandem-repeat type lectins. Here, rSmLgals3 strong binding ability to LPS and PGN,
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followed by LTA. In Crassostrea gigas, galectin-3 showed the highest affinity to PGN,
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as well as Gram-positive bacteria, Gram-negative bacteria and fungi [13]. However,
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galectin-2 (proto type) did not bind to LPS, PGN, or glucan, while it bound
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ACCEPTED MANUSCRIPT Gram-negative bacteria and fungi in Crassostrea gigas [13]. In addition, recombinant
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galectin-3 can also function like a chemokine to induce the migration of monocytes
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and macrophages in human [47]. As serving as a bridge between the cell and the
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extracellular matrix to promote adhesion, recombinant galectin-3 could increase the
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adhesive properties of human neutrophils to laminin [48].
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Notably, galectin-3 possesses a unique feature to oligomerize after glycoprotein
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binding at the cell surface, with the N terminus self-association domain [48]. In this
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regard, galectin-3 has been reported to influence the stability of cell-cell adhesion
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matrix to involve in many biological processes with other genes [49]. Especially,
384
galectin-3 was reported to contribute to epithelial barrier integrity. For example,
385
galectin-3 could regulate the barrier function of the intestinal epithelium by
386
controlling Desmoglein-2 protein stability and intercellular adhesion [50]. In gastric
387
epithelial cells, knock down of galectin-3 led to reduced NF-κB promoter activity and
388
interleukin-8 (IL-8) secretion, suggesting its pro-inflammatory roles [41]. In airway
389
epithelial cells, galectin-3 was reported to regulate inflammatory response by
390
modulating the expression of SOCS1 and RIG1 [51]. Taken together, galectin-3 plays
391
vital roles in mucosal homeostasis and barrier maintenance. However, the knowledge
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of galectin-3 are still limited in teleost species, further studies should be carried out to
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better characterize its detailed roles in teleost mucosal immunity.
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Acknowledgement
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This study was supported by the National Science Foundation of China
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(No.:31602193). And it was also supported by keypoint research and invention
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program in Shandong Province (2017GHY215004).
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and their ligands: amplifiers, silencers or tuners of the inflammatory response?, Trends in Immunology 23(6) (2002) 313-320.
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Scophthalmus maximus (L.), intestine a portal of entry for the fish pathogen Vibrio anguillarum?, [36] Olsson, Jöborn, Westerdahl, Blomberg, Kjelleberg, Conway, Survival, persistence and Journal of Fish Diseases 21(1) (2010) 1-9.
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proliferation of Vibrio anguillarum in juvenile turbot, Scophthalmus maximus (L.), intestine and faeces, [37] R. O'Toole, H.J. Von, R. Rosqvist, P.E. Olsson, H. Wolfwatz, Visualisation of zebrafish infection by GFP-labelled Vibrio anguillarum, Microbial Pathogenesis 37(1) (2004) 41-46. [38] X. Liu, H. Wu, X. Chang, Y. Tang, Q. Liu, Y. Zhang, Notable mucosal immune responses induced in the intestine of zebrafish ( Danio rerio ) bath-vaccinated with a live attenuated Vibrio anguillarum vaccine, Fish & Shellfish Immunology 40(1) (2014) 99-108.
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Japanese Flounder Paralichthys olivaceus, Fish Pathology 36(3) (2009) 40-41. [40] C. Li, R. Wang, B. Su, Y. Luo, J. Terhune, B. Beck, E. Peatman, Evasion of mucosal defenses during Aeromonas hydrophila infection of channel catfish ( Ictalurus punctatus ) skin, Developmental & Comparative Immunology 39(4) (2013) 447-455. [41] V.V. Subhash, B. Ho, Galectin 3 acts as an enhancer of survival responses in H. pylori-infected
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gastric cancer cells, Cell Biology & Toxicology 32(1) (2016) 23-35. [42] H.F. Tsai, C.S. Wu, Y.L. Chen, H.J. Liao, I.T. Chyuan, P.N. Hsu, Galectin-3 suppresses mucosal inflammation and reduces disease severity in experimental colitis, Journal of Molecular Medicine 94(5) (2016) 545-556.
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[43] E. Altman, B.A. Harrison, R.K. Latta, K.K. Lee, J.F. Kelly, P. Thibault, Galectin-3-mediated adherence of Proteus mirabilis to Madin-Darby canine kidney cells, Biochemistry & Cell Biology-biochimie Et Biologie Cellulaire 79(6) (2001) 783-788. [44] M. Fowler, R.J. Thomas, J. Atherton, I.S. Roberts, N.J. High, Galectin-3 binds to Helicobacter pylori O-antigen: it is upregulated and rapidly secreted by gastric epithelial cells in response to H-pylori adhesion, Cellular Microbiology 8(1) (2006) 44–54. [45] R.S. Gupta, V. Bhandari, Phylogeny and molecular signatures for the phylum Thermotogae and its subgroups, Antonie Van Leeuwenhoek 100(1) (2011) 1-34. [46] P. Quattroni, Y. Li, D. Lucchesi, S. Lucas, D.W. Hood, M. Herrmann, H.J. Gabius, C.M. Tang, R.M. Exley, Galectin-3 binds Neisseria meningitidis and increases interaction with phagocytic cells, Cellular Microbiology 14(11) (2012) 1657-1675. [47] R.Y. Yang, G.A. Rabinovich, F.T. Liu, Galectins: structure, function and therapeutic potential,
ACCEPTED MANUSCRIPT [48] I. Kuwabara, F.T. Liu, Galectin-3 promotes adhesion of human neutrophils to laminin, Journal of Immunology 156(10) (1996) 3939-3944. [49] H. Inohara, A. Raz, Functional evidence that cell surface galectin-3 mediates homotypic cell adhesion, Cancer Research 55(15) (1995) 3267. [50] K. Jiang, C.R. Rankin, P. Nava, R. Sumagin, R. Kamekura, S.R. Stowell, M. Feng, C.A. Parkos, A. Biological Chemistry 289(15) (2014) 10510-7.
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Nusrat, Galectin-3 regulates desmoglein-2 and intestinal epithelial intercellular adhesion, Journal of [51] N.L. Mihai, B. Aditi, F. Chiguang, G.R. Vasta, Galectins regulate the inflammatory response in airway epithelial cells exposed to microbial neuraminidase by modulating the expression of SOCS1
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and RIG1, Molecular Immunology 68(2) (2015) 194-202.
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Expert Reviews in Molecular Medicine 10(article e17) (2015) e17.
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Table 1. Primers used in this study
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Primer
Sequence(5’-3’)
qRT-PCR
5’AGGACTGTGAGCGGCTATAC3’
Sm-lgals3 F
5’ACAAAGCCGTACGAGAGACA3’
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Sm-lgals3 R
5’ATGGCCGTTCTTAGTTGGTG3’
18s RNA F
5’CTCAATCTCGTGTGGCTGAA3’
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18s RNA R Protein expression
5’ATGGCGGATTTCTCGCTGAC3’
Sm-lgals3-pr F
5’TCAGATCATGCTCGGGGC3’
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584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607
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Sm-lgals3-pr R
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Analysis
Lgals3
No. of amino acids
400
Molecular weight (kDa)
39.74
Theoretical pI
5.12
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Total number of negatively charged residues 24 (Asp + Glu) Total number of positively charged residues 13 (Arg + Lys) Formula
C1814H2637N477O516S11
Instability index
54.9
Aliphatic index
45.42
0
Casein kinase II phosphorylation site
1
N-glycosylation site
0
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Protein kinase C phosphorylation site
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619 620 621 622 623 624 625 626 627 628 629 630 631 632
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Grand average of hydropathicity (GRAVY) -0.354
617 618
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Table 2. Primary structural analysis. Properties of turbot SmLgals3 genes determined by ProtParam.
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608 609 610 611 612 613 614 615 616
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Table 3. Amino acid comparison of SmLgals3 genes with other species using MatGAT program.
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Turbot lgals3
Species
Similarity
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Turbot lgals3 Human lgals3
Mouse lgals3 Chicken lgals3 Tetraodon lgals3
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Medaka lgals3
Large yellow croaker lgals3
642 643 644 645 646 647 648 649 650 651 652 653 654 655 656
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641
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Zebrafish lgals3
43.8
35.1
49.3
38.7
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Green Turtle lgals3
Tilapia lgals3
Identity
45
36
42
35.7
62.3
58.4
66.3
61.3
64.3
60.1
68
56.3
66.5
58.3
ACCEPTED MANUSCRIPT 657 658 659 660 661 662
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663 664
Fig. 1 Exon/intron organization of SmLgals3 gene was obtained by using Splign to
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align their cDNA sequences to the turbot genome. Boxes indicate exons and dashes
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indicate introns. The dark shaded boxes indicate exon sequences that encoding amino
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acids.
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669 670 671 672 673 674 675 676 677 678 679 680 681
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665
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Fig. 2. Phylogenetic tree for the SmLgals3 gene. The phylogenetic tree was constructed based on the amino acid sequences of SmLgals3 from different species using the neighbor-joining method in MEGA 6. Gaps were removed by complete deletion and the phylogenetic tree was evaluated with 1,000 bootstrap replications. The bootstrapping values were indicated by numbers at the nodes. Dark solid circles indicated the newly characterized SmLgals3 genes.
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699 700 701 702 703 704 705 706 707 708 709 710 711 712 713
AC C
698
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682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697
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Fig. 3. Syntenic analysis of SmLgals3 genes from turbot, human, chicken, tetraoden, zebrafish, medaka, spotted gar, fugu. The Lgals3 gene is highlighted by bright blue color filled boxes. CDKN3: Cyclin-dependent kinase inhibitor 3; CNIH1: cornichon family AMPA receptor auxiliary protein 1; GMFB: Glia maturation factor beta; L2HGDH: L-2-hydroxyglutarate dehydrogenase; MED6: mediator complex subunit 6; SOS2: Ras/Rho guanine nucleotide exchange factor 2; ACTN1:actinin alpha 1.
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714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730
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Fig. 4. The tissue distribution of SmLgals3 in turbot. SmLgals3 expression in liver, skin, spleen, blood, head kidney, intestine, gill and brain was determined by quantitative real-time PCR. The expression level of SmLgals3 in skin was set as 1. The relative abundance of SmLgals3 was expressed as mean ± SE (N = 3).
765 766 767 768 769 770 771 772
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760 761 762 763 764
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749 750 751 752 753 754 755 756 757 758 759
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Fig. 5. The expression patterns of SmLgals3 in turbot tissues at different timepoints (2h, 6h, 12h, and 24h) after V. anguillarum challenge. SmLgals3 expression in skin, intestine and gill was determined by quantitative real-time PCR. 18S rRNA was employed as an internal control. Asterisks (*) marked the significant differences between experimental and control groups (P < 0.05). Error bars indicated standard error (n = 3).
790 791 792 793 794 795 796 797
AC C
EP
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784 785 786 787 788 789
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773 774 775 776 777 778 779 780 781 782 783
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Fig. 6. The expression patterns of SmLgals3 in turbot mucosal tissues at different timepoints (2h, 4h, 8h, and 12h) after S. iniae challenge. SmLgals3 expression in skin, intestine and gill was determined by quantitative real-time PCR. 18S rRNA was employed as an internal control. Asterisks (*) marked the significant differences between experimental and control groups (P < 0.05). Error bars indicated standard error (n = 3).
AC C
EP
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804 805 806 807 808 809 810 811 812 813 814 815
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798 799 800 801 802 803
816 817 818 819 820 821 822
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Fig. 7. Results of the in vitro binding assay of SmLgals3 on microbial ligands, including lipopolysaccharide, peptidoglycan, and lipoteichoic acid. * indicate a significant difference in the absorbance between different microbial ligands that exposed to r SmLgals3 and the control group: *p < 0.05; **p < 0.01.
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823 824 825 826 827 828 829 830 831 832
834
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833
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SmLgals3 gene is homologous to their counterparts in other vertebrates. SmLgals3 gene was ubiquitously expressed in turbot tissues.
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SmLgals3 had strong binding ability to LPS, LTA, and PGN.
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SmLgals3 gene showed different expression patterns following different bacterial challenge.
S1050-4648(18)30633-8
DOI:
10.1016/j.fsi.2018.10.009
Reference:
YFSIM 5610
To appear in:
Fish and Shellfish Immunology
Received Date: 4 August 2018 Revised Date:
12 September 2018
Accepted Date: 5 October 2018
Please cite this article as: Tian M, Yang N, Zhang L, Fu Q, Tan F, Li C, Expression profiling and functional characterization of galectin-3 of turbot (Scophthalmus maximus L.) in host mucosal immunity, Fish and Shellfish Immunology (2018), doi: https://doi.org/10.1016/j.fsi.2018.10.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Expression profiling and functional characterization of
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galectin-3 of turbot (Scophthalmus maximus L.) in host
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mucosal immunity
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Mengyu Tiana,1, Ning Yang a,1, Lu Zhanga, Qiang Fua, Fenghua Tana, Chao Lia,*,
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266109, People’s Republic of China
Marine Science and Engineering College, Qingdao Agricultural University, Qingdao
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a
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*
All these authors contributed equally to this work.
Corresponding author: Chao Li, [email protected]
ACCEPTED MANUSCRIPT Abstract: Galectins, a family of evolutionary conserved β-galactoside-binding
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proteins, have been characterized in wide range of species. Galectin-3 is the only
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member in the chimera type, which is monomeric lectin with one CRD domain. A
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growing body of evidence have indicated vital roles of galectin-3 in innate immune
29
responses against infection. Here, one galectin-3 gene was captured in turbot
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(SmLgals3) contains a 1,203 bp open reading frame (ORF). In comparison to other
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species, SmLgals3 showed the highest similarity and identity to large yellow croaker
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and medaka, respectively. The genomic structure analysis showed that SmLgals3 had
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5 exons similar to other vertebrate species. The syntenic analysis revealed that
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galectin-3 had the same neighboring genes across all the selected species, which
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suggested the synteny encompassing galectin-3 region during vertebrate evolution.
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Subsequently, SmLgals3 was widely expressed in all the examined tissues, with the
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highest expression level in brain and the lowest expression level in skin. In addition,
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SmLgals3 was significantly down-regulated in intestine following both Gram-negative
39
bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae
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immersion challenge. Finally, the rSmLgals3 showed strong binding ability to all the
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examined microbial ligands. Taken together, our results suggested SmLgals3 plays
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vital roles in fish innate immune responses against infection. However, the knowledge
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of SmLgals3 are still limited in teleost species, further studies should be carried out to
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better characterize its detailed roles in teleost mucosal immunity.
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Keyword: Galectin-3; turbot; Vibrio anguillarum; Streptococcus iniae; microbial
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ligand binding
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1. Introduction
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Galectins, a family of evolutionary conserved β-galactoside-binding proteins, have been characterized in a wide range of species, including fungi, parazoa, and
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vertebrates [1, 2]. The galectins were featured with a characteristic carbohydrate
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recognition domain (CRD) with an affinity for β-galactosides, and a conserved
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sequence motif [3]. According to the structural features, galectin family has been
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classified into three different types: proto-type, Chimera type and tandem-repeat type
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[4]. As a group of carbohydrate-binding proteins, galectins could involve in many
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biological processes, such as immune responses, inflammation, cell migration and
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development [5]. So far, fourteen galectin members have been discovered in
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mammals, but lower vertebrates and invertebrates appear to have a smaller number of
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galectins. In teleost, several studies have been carried out to characterize galectin
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genes, since the first galectin gene was identified in electric organs of the electric eel
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(Electrophorus electricus) [6]. For instance, eight galectins were identified in catfish,
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and showed significant expression changes following Edwardsiella ictaluri and
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Flavobacterium columnare challenge in mucosal tissues [7].
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Among three galectin types, only galectin-3 belongs to the chimera type, which are
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monomeric lectins with one CRD domain. Similar to the other galectin types,
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galectin-3 also lacks a signal sequence which is required for secretion, but the
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galectin-3 protein could be released into the extracellular space [8]. Of note,
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galectin-3 has been captured in a wide range of immune cells, including mast cells,
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macrophages, neutrophils, monocytes and T cells. For instance, galectin-3 deletion
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suppressed production of pro-inflammatory cytokines and significantly reduced
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activation of NOD-like receptor family in macrophages in mice [9]. In T cells,
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and Th2 cells [11]. In teleost species, galectin-3 could reduce the adhesion of
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infectious hematopoietic necrosis virus by direct interaction with virus envelop on the
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epithelial cell surface in a carbohydrate-dependent manner [12]. In oyster
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(Crassostrea gigas), galectin-3 displayed agglutination ability against both
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Micrococcus luteus and Pichia pastoris [13]. However, most of the studies of
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galectin-3 were performed in mammals, the characterization of galectin-3 in teleost
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was still limited in a handful species.
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Turbot (Scophthalmus maximus L.), one of the most extensively maricultured
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species in China, suffers from the bacterial disease including Vibrio anguillarum and
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Streptococcus iniae. Many studies have been performed to identify the
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immune-related genes and investigate their associated activities during bacterial
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infection [14-18], for further development of the disease control and prevention
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measures in turbot. In this regard, we sought here to characterize galectin-3 gene in
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turbot, and its expression patterns following different bacterial infection, as well as
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microbial ligand-binding activities to gain initial insight into the immune roles of
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galectin-3 in turbot.
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2. Materials and methods
2.1. Sequence identification and analysis
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The turbot galectin-3 gene was captured from transcriptome database [19] by
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BLAST program using galectin-3 protein sequences from other species as queries.
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The retrieved candidate sequences were then translated using ORF Finder
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further verified against NCBI non-redundant protein sequence database by BLASTP
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(http://blast.ncbi.nlm.nih.gov/Blast.cgi). The conserved domains and signal peptides
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were further identified by the simple modular architecture research tool (SMART;
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http://smart.embl-heidelberg.de/). The theoretical pI, molecular mass and
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N-glycosylation sites were captured in ExPASy server [20]. The intron and exon
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structures were predicted by Splign program [21].
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2.2. Phylogenetic analysis
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The phylogenetic tree was constructed based on the amino acid sequences of galectin-3 gene from various species including human (Homo sapiens), shrew mouse
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(Mus pahari), chicken (Gallus gallus), green sea turtle (Chelonia mydas), spotted gar
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(Lepisosteus oculatus), zebrafish (Danio rerio), medaka (Oryzias latipes), Tilapia
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(Oreochromis niloticus), fugu (Takifugu rubripes). The multiple protein sequence
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alignment was performed in Clustal Omega program [22]. A neighbor-joining
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phylogenetic tree was generated using the Molecular Evolutionary Genetics Analysis
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(MEGA 6) [23].
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2.3. Syntenic analysis
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Syntenic analysis was performed to further verify the identification of turbot
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galectin-3 gene. Briefly, the protein sequences of neighboring genes of the turbot
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galecin-3 were predicted from the turbot scaffold using FGENESH program. The
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identified protein sequences were annotated by BLASTP program against NCBI
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non-redundant (nr) database. The conserved syntenic pattern of galectin-3 gene in
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other species were characterized in Ensembl database and Genomicus [24].
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2.4. Bacteria challenge and sample collection
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In order to characterize the roles of galectin-3 gene in the host immune responses against bacterial infection, the Gram-negative bacteria V. anguillarum and
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Gram-positive bacteria S. iniae were selected to conduct the immersion challenge.
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Turbot fingerlings (average body weight: 15.6 g and average body length = 5.5 cm)
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were obtained from the turbot hatchery (Haiyang, Shandong, China), and acclimated
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in the laboratory in a flow-through system for at least two weeks prior to challenge.
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After a pre-challenge, the bacteria was re-isolated from single symptomatic fish and
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biochemically confirmed before experiment. During the experiment, the experimental
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fish were challenged for 2 h by immersion, and then transferred in fresh water. At
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each timepoint following challenge, skin, gill and intestine samples were collected
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from 15 fish (5 fish per pool) from the appropriate aquaria.
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Briefly, the V. anguillarum was inoculated in LB broth in a shaker (180 rpm) at
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28 °C overnight. The fish were immersed at a final concentration of 5×107 CFU/mL
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for 2 h, while the control fish were immersed in sterilized media alone. Aquaria were
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randomly assigned for 2 h, 6 h, 12 h and 24 h post-treatment and 0 h control with
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thirty fish in each aquarium for sample collection.
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The S. iniae isolate was inoculated in LB medium in a shaker incubator at 28 °C
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overnight. The fish were equally divided into five aquariums, 4 challenged groups (2
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h, 4 h, 8 h and 12 h) and one control group. For the challenge, the fish were immersed
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for 2 h at a final concentration of 5×106 CFU/mL, while the control fish were
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immersed in sterilized media alone. All samples from both experiments were
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flash-frozen in liquid nitrogen and then stored in a -80 °C ultra-low freezer until
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preparation of RNA.
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2.5. Total RNA extraction and cDNA synthesis
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Prior to RNA extraction, tissue samples were homogenized under liquid nitrogen
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using mortar and pestle. Total RNA was extracted using Trizol® Reagent (Invitrogen,
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USA) according to the supplied protocol. The quality and quantity of RNA of each
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sample were measured on a Nanodrop 2000 (Thermo Electron North America LLC,
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FL). All extracted samples had an A260/280 ratio greater than 1.8, and were diluted to
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250 ng/µl.
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2.6. Real-time PCR analysis
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Gene specific primers were designed using Primer3 software based on the turbot galectin-3 sequences, and 18S rRNA gene was used as a reference gene (Table 1).
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First strand cDNA was synthesized by PrimeScript RT reagent Kit (TaKaRa)
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according to manufacturer’s protocol (500 ng RNA per 10 µl reaction). Quantitative
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real-time PCR (qPCR) was performed on a CFX96 real-time PCR detection system
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(Bio-Rad Laboratories, Hercules, CA) using the SYBR ExScript qRT-PCR Kit
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(Takara, Dalian, China). The reaction systems for all real-time PCR were as follows:
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1.0 µL of each primer (5 µM), 5.0 µL SYBR Green supermix, 2.0 µL
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RNase/DNase-free water, and 1.0 µL 200 ng/µL cDNA. The PCR reaction mixture
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was denatured at 95 °C for 30 s and then subjected to 40 cycles of 95 °C for 5 s, 58 °C
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ACCEPTED MANUSCRIPT for 5 s and followed by dissociation curve analysis, 5 s at 65 °C, then up to 95 °C at a
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rate of 0.1 °C/s increment, to verify the specificity of the amplicons. Results were
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analyzed using Relative Expression Software Tool (REST) to capture the significance
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at the level of P<0.05 [25]. In order to determine the gene expression patterns in
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turbot healthy tissues, the tissue with the lowest Ct values were used as control. The
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mRNA expression levels of all samples were normalized to the levels of 18S
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ribosomal RNA gene in the same samples. A no-template control was run on all
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plates.
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2.7. Plasmid construction
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In order to construct the expression plasmid for SmLgals3, SmLgals3 was amplified with the specific primers following cDNA synthetization. After gel extraction, the
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PCR products were ligated to pEASY-Blunt-E1 vector, and then transformed into
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competent Trans1-T1 cells. Following blue-white spotting selection, the positive
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clones were selected and sequencing with T7 Promoter Primer. The verified
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recombinant plasmid was extracted and marked as pEASY-E1- Lgals3.
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2.8. Expression and purification of recombinant SmLgals3
The recombinant plasmid pEASY-E1- Lgals3 was transformed into E.coli BL21
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(DE3). The transformant BL21- Lgals3 and the control BL21 with empty pET-32a
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were cultured in LB medium, and induced by adding 0.5 mM
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isopropyl-b-D-thiogalactopyranoside. The expressed protein was purified by
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nickel-nitrilotriacetic acid chromatography, and analyzed by 12% sodium dodecyl
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sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by staining
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with Coomassie brilliant blue. The concentration of the recombinant protein was
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determined using Bradford's method.
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2.9. Solid-phase enzyme-linked immunosorbent assay (ELISA)
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The binding ability of rSmLgals3 with lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN) was detected by ELISA method. Briefly, LPS/LTA
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/PGN (5 µg/ml) were coated to 96 microtitor plate at 4 °C overnight. The wells were
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washed with 300 µl PBST three times, and then blocked with 100 µl 5% BSA at 4 °C
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for 1 h. Then, 100 µl of increasing concentrations of purified recombinant SmLgals3
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(0.5, 1, 2, 4, 8 and 16 µg/mL) were added into each ligand-coated well, with 4
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replicates for each concentration, and incubated at 37 °C for 1h. Subsequently, the
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wells were incubated at 37 °C for 1 h with 100 µl mouse anti-His antibody (Solarbio,
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Beijing, China) (diluted 1:1000 in 5% BSA), followed by another incubation at 37 °C
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for 40 min with the addition of 100 µl horseradish peroxidase conjugated goat
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anti-mouse IgG (Solarbio, Beijing, China) (diluted 1:1000 in 5% BSA). Finally, the
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reaction was terminated by adding 0.5M sulfate, and the plate was then read at 450
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nm with an ELISA reader.
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3. Results
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3.1 Identification of turbot galectin-3 genes
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After searching the turbot transcriptome and genome database [26] using available
ACCEPTED MANUSCRIPT galectin-3 sequences from other species as queries, one galectin-3 gene was captured
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in turbot (SmLgals3). In detail, the full-length SmLgals3 (GenBank accession:
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MF797865) transcript contains a 1,203 bp open reading frame (ORF) encoding 400
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amino acids residues with a predicted molecular mass of a 39.74 kDa and a theoretical
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isoelectric point of 5.12 (Table 2). The deduced SmLgals3 protein was predicted to
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have 1 Casein kinase II phosphorylation sites, as well as 24 negatively charged
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residues (Asp + Glu), 13 positively charged residues (Arg + Lys), and with an
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instability index of 54.9 and aliphatic index of 45.42 (Table 2). In comparison to other
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species, SmLgals3 showed the highest similarity to large yellow croaker (68%),
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followed by zebrafish (66.5%), while the highest identity was observed with medaka
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(61.3%), followed by tilapia (60.1%) (Table 3).
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3.2. Genomic structure analysis of SmLgals3 gene
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Following sequence identification, the genomic architecture of SmLgals3 was also investigated. Following the comparison of the exon/intron organization of
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SmLgals3 with other vertebrates, the N-terminal and C-terminal showed different
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similarities among the selected species. In detail, the exon number was varied from 5
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to 7, and the CRD domain was composed by three same exons (89 bp,166 bp and 156
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bp) in all the species (Fig.1), whereas the different number of exons were observed in
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their N-terminal domains. In general, there were 5 exons in the mammals and chicken,
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and more exons (6 and 7) in fish species, except turbot also showed 5 exons (Fig.1).
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3.3. Phylogenetic analyses
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Subsequently, the phylogenetic analysis was performed with amino acid sequences of galectin-3 proteins from species of fish and mammals, using the
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neighbor-joining method in MEGA 6. In our results, SmLgals3 was firstly clustered
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with fugu, and then clustered with tilapia, medeka, and zebrafish (Fig. 2). The higher
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vertebrates formed single clade including human, mouse, chicken and green sea turtle
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(Fig. 2). And all branching nodes were supported by high bootstrap values.
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3.4. Syntenic analysis
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Finally, the syntenic analysis was performed for further validation of the
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identification of SmLgals3 gene. In general, a relatively conserved synteny was
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observed in comparison of SmLgals3 to other species. After searching the genomic
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databases of different species, the same neighboring genes were found across the
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selected species, including CDKN3, CNIH1, GMFB, L2HGDH, MED6, SOS2, and
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ACTN1 (Fig. 3).
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3.5. The tissue distribution of SmLgals3
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The tissue distribution of SmLgals3 was examined in eight turbot healthy tissues by
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real-time PCR method. In our results, SmLgals3 was widely expressed in all the
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examined tissues, with the highest expression level in brain (46.28 fold), followed by
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intestine (12.6 fold), head-kidney (7.15 fold) and blood (6.2 fold), while the lowest
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expression level was detected in skin (Fig. 4).
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3.6. Expression profiles of SmLgals3 following bacterial challenge
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In order to characterize the immune roles of SmLgals3 in turbot mucosal immunity, the expression profiles of SmLgals3 were investigated in mucosal tissues (gill, skin
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and intestine) at early timepoints following immersion challenge with Gram-negative
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bacteria V. anguillarum, and Gram-positive bacteria S. iniae, respectively.
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Following V. anguillarum challenge, SmLgals3 was significantly down-regulated in all the three tissues except up-regulated in skin with 12.36 fold at 2 h, but it was
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quickly down-regulated with -4.65 fold at 6 h, and then returned to basal level (Fig. 5).
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The most up-regulation was detected in intestine at 6 h with -28.19 fold, and -6.59
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fold at 12 h. Different from other tissues, the gill showed -3.58 fold at 6 h, -9.64 fold
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at 12 h, and the most dramatic down-regulation at 24 h with -22.99 fold (Fig. 5).
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In S. iniae challenge, the SmLgals3 was down-regulated much more quickly than that in V. anguillarum challenge. The SmLgals3 was down-regulated at 2h at all the
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three tissues, with -13.01 fold in intestine, -10.78 fold in skin, and -3.43 fold in gill
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(Fig. 6). Interestingly, it quickly returned to basal level in gill and skin, while continue
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to be down-regulated in intestine with -9.58 fold at 8h and -15.46 fold at 12h (Fig. 6).
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3.7. Microbial ligand-binding in vitro
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The binding ability of SmLgals3 to microbial ligands was also investigated to
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further characterize its function. Briefly, the rSmLgals3 was purified from E. coli as a
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native His-tagged protein. In SDS-PAGE analysis, only a single band was observed
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(Supplementary Fig. 1). In our results, the strong binding ability was observed to LPS,
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PGN and LTA (Fig. 7). The strongest binding ability was detected to LPS which
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showed significant binding ability at 0.5µg/ml, while the significant binding ability
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for PGN was observed at 0.5µg/ml, and 2.0 µg/ml for LTA (Fig. 7).
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4. Discussion
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Galectins are widely distributed in primary and secondary lymphoid organs and
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many types of immune cells, with vital roles in regulation of immune cell
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proliferation and apoptosis [27]. A growing body of evidence have indicated
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galectin-3 could involve in chemotaxis, phagocytosis, LPS-induced IL-1 production,
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NADPH-oxidase activity, and IL-4 induced survival of memory B cells [28-30].
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Although considerable experimental evidences have elucidated galectin functions
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within the immune system in higher mammals, the characterization of galectin-3 is
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still lacking in teleost species. Here, we identified galectin-3 gene in turbot,
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performed expression profiling analysis of galectin-3 in response to different bacterial
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infection, as well as the binding ability analysis to different microbial ligands, to
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provide initial information for further functional characterization of galectin-3 in
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teleost. In this study, one galectin-3 gene was captured in turbot (SmLgals3) with
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similar molecular properties to other fish species (Table 1). As an evolutionarily
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highly conserved family of proteins, the genomic structure analysis, phylogenetic
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analysis and synteny analysis validated the identification of SmLgals3 and showed the
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strong orthology to their counterparts vertebrate species.
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In tissue distribution analysis, SmLgals3 was widely expressed in all the examined
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tissues, with the highest expression level in brain, followed by intestine (Fig. 4).
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Interestingly, galectin-3 was reported to be widely expressed in rat brain, and seems to
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be regulated by developmental cascades [31], also was considered as mediator of
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microglia responses in injured brain [32]. In addition, galectin-3 was highly expressed
ACCEPTED MANUSCRIPT in the colons of patients with ulcerative colitis [33]. However, galectin-3 was highly
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expressed in gill and skin, and lowly expressed in intestine [34], while it showed the
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lowest . With the limited information about expression profiling of galectin-3 in
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teleost, the putative roles of galectin-3 in different tissues of teleost need to be further
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characterized.
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Subsequently, the expression patterns of SmLgals3 were characterized following
different bacteria challenge in order to gain insight into its immune roles in mucosal
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immunity. Notably, SmLgals3 showed significant down-regulation in all the tissues
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following both Gram-negative bacteria V. anguillarum, and Gram-positive bacteria S.
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iniae challenge, with the most down-regulation in intestine. Previously, V.
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anguillarum cells were detected in spleens from more than 50% of orally or rectally
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infected fish, suggesting that the intestine might serving as a portal of entry for V
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anguillarum [35]. Following orally administered V. anguillarum in turbot, the bacteria
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showed strong ability to survive in the acidic environment of the stomach, and persist
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in the intestine, which suggested that the turbot intestinal tract could serve as an
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enrichment site for V. anguillarum [36]. In zebrafish, the entry of V. anguillarum
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through the mouth into the intestine was detected at 2h following bath challenge with
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V. anguillarum, and the localisation of V. anguillaum was detected in intestine at 6h
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[37]. Moreover, following bath-vaccination of live attenuated V. anguillarum vaccine,
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bacteria cells proliferated rapidly in 3h after vaccination and maintained at a high
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level until 6h in the intestine, with a significant up-regulation of TLR5 triggering
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MyD88-dependent signaling pathway in the intestine [38]. Comparing to V.
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anguillarum challenge, SmLgals3 was significantly down-regulated in all the
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timepoints in the intestine following S. iniae challenge. In Japanese flounder, the S.
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iniae bath challenge could induce much more significant deaths in lower
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ACCEPTED MANUSCRIPT concentration than oral challenge [39]. Previously, several immune-related genes were
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also significantly up-regulated following S. iniae infection in turbot intestine [14-16].
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In contrast, galectin-3 was significantly up-regulated in catfish skin following
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Aeromonas hydrophila infection [40]. Although our knowledge of galectin-3 functions
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are limited in fish, it has been well documented in mammalian species. For instance,
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In gastric epithelial cells, galectin-3 was up-regulated following Helicobacter pylori
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infection [41]. Ggalectin-3 showed the inhibition effect on the colonic mucosa
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inflammation and reduced disease severity by inducing regulatory T cells [42].
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Galectin-3 deletion resulted in the suppressed production of pro-inflammatory
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cytokines in colonic macrophages and favored their alternative activation, as well as
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significantly reducing activation of NOD-like receptor family in macrophages [33].
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Several studies following different bacteria challenge, suggested that galectin-3 may
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act as a cell surface docking site or a cross-linking molecule promoting adhesion
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[43-46]. Further work is warranted to examine whether turbot galectin-3 may play
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similar roles in supporting pathogen adhesion.
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As typical lectins, galectins possessed carbohydrate binding ability to microbial
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pathogens by recognizing exogenous ligands, especially carbohydrates on the surface
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of microbes via their CRDs, and subsequently trigger downstream immune signaling
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pathways to eliminate the pathogens. The structure features might affect their
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specificity and binding affinity to carbohydrates. As the only member of chimera type
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galectins, galectin-3 should have different immune functions compared to proto and
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tandem-repeat type lectins. Here, rSmLgals3 strong binding ability to LPS and PGN,
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followed by LTA. In Crassostrea gigas, galectin-3 showed the highest affinity to PGN,
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as well as Gram-positive bacteria, Gram-negative bacteria and fungi [13]. However,
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galectin-2 (proto type) did not bind to LPS, PGN, or glucan, while it bound
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ACCEPTED MANUSCRIPT Gram-negative bacteria and fungi in Crassostrea gigas [13]. In addition, recombinant
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galectin-3 can also function like a chemokine to induce the migration of monocytes
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and macrophages in human [47]. As serving as a bridge between the cell and the
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extracellular matrix to promote adhesion, recombinant galectin-3 could increase the
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adhesive properties of human neutrophils to laminin [48].
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Notably, galectin-3 possesses a unique feature to oligomerize after glycoprotein
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binding at the cell surface, with the N terminus self-association domain [48]. In this
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regard, galectin-3 has been reported to influence the stability of cell-cell adhesion
383
matrix to involve in many biological processes with other genes [49]. Especially,
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galectin-3 was reported to contribute to epithelial barrier integrity. For example,
385
galectin-3 could regulate the barrier function of the intestinal epithelium by
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controlling Desmoglein-2 protein stability and intercellular adhesion [50]. In gastric
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epithelial cells, knock down of galectin-3 led to reduced NF-κB promoter activity and
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interleukin-8 (IL-8) secretion, suggesting its pro-inflammatory roles [41]. In airway
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epithelial cells, galectin-3 was reported to regulate inflammatory response by
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modulating the expression of SOCS1 and RIG1 [51]. Taken together, galectin-3 plays
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vital roles in mucosal homeostasis and barrier maintenance. However, the knowledge
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of galectin-3 are still limited in teleost species, further studies should be carried out to
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better characterize its detailed roles in teleost mucosal immunity.
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Acknowledgement
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This study was supported by the National Science Foundation of China
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(No.:31602193). And it was also supported by keypoint research and invention
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program in Shandong Province (2017GHY215004).
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[10] D.K. Hsu, S.R. Hammes, I. Kuwabara, W.C. Greene, F.T. Liu, Human T lymphotropic virus-I infection of human T lymphocytes induces expression of the beta-galactoside-binding lectin, galectin-3, American Journal of Pathology 148(5) (1996) 1661-70. [11] M.A. Toscano, G.A. Bianco, J.M. Ilarregui, D.O. Croci, J. Correale, J.D. Hernandez, N.W. Zwirner, F. Poirier, E.M. Riley, L.G. Baum, Differential glycosylation of TH1, TH2 and TH-17 effector cells
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Nusrat, Galectin-3 regulates desmoglein-2 and intestinal epithelial intercellular adhesion, Journal of [51] N.L. Mihai, B. Aditi, F. Chiguang, G.R. Vasta, Galectins regulate the inflammatory response in airway epithelial cells exposed to microbial neuraminidase by modulating the expression of SOCS1
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and RIG1, Molecular Immunology 68(2) (2015) 194-202.
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543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575
Expert Reviews in Molecular Medicine 10(article e17) (2015) e17.
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532 533 534 535 536 537 538 539 540 541 542
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Table 1. Primers used in this study
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576 577 578 579 580 581 582 583
Primer
Sequence(5’-3’)
qRT-PCR
5’AGGACTGTGAGCGGCTATAC3’
Sm-lgals3 F
5’ACAAAGCCGTACGAGAGACA3’
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Sm-lgals3 R
5’ATGGCCGTTCTTAGTTGGTG3’
18s RNA F
5’CTCAATCTCGTGTGGCTGAA3’
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18s RNA R Protein expression
5’ATGGCGGATTTCTCGCTGAC3’
Sm-lgals3-pr F
5’TCAGATCATGCTCGGGGC3’
EP AC C
584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607
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Sm-lgals3-pr R
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Analysis
Lgals3
No. of amino acids
400
Molecular weight (kDa)
39.74
Theoretical pI
5.12
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Total number of negatively charged residues 24 (Asp + Glu) Total number of positively charged residues 13 (Arg + Lys) Formula
C1814H2637N477O516S11
Instability index
54.9
Aliphatic index
45.42
0
Casein kinase II phosphorylation site
1
N-glycosylation site
0
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Protein kinase C phosphorylation site
AC C
619 620 621 622 623 624 625 626 627 628 629 630 631 632
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Grand average of hydropathicity (GRAVY) -0.354
617 618
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Table 2. Primary structural analysis. Properties of turbot SmLgals3 genes determined by ProtParam.
SC
608 609 610 611 612 613 614 615 616
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Table 3. Amino acid comparison of SmLgals3 genes with other species using MatGAT program.
RI PT
633 634 635 636 637 638 639 640
Turbot lgals3
Species
Similarity
SC
Turbot lgals3 Human lgals3
Mouse lgals3 Chicken lgals3 Tetraodon lgals3
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Medaka lgals3
Large yellow croaker lgals3
642 643 644 645 646 647 648 649 650 651 652 653 654 655 656
AC C
641
EP
Zebrafish lgals3
43.8
35.1
49.3
38.7
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Green Turtle lgals3
Tilapia lgals3
Identity
45
36
42
35.7
62.3
58.4
66.3
61.3
64.3
60.1
68
56.3
66.5
58.3
ACCEPTED MANUSCRIPT 657 658 659 660 661 662
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663 664
Fig. 1 Exon/intron organization of SmLgals3 gene was obtained by using Splign to
666
align their cDNA sequences to the turbot genome. Boxes indicate exons and dashes
667
indicate introns. The dark shaded boxes indicate exon sequences that encoding amino
668
acids.
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669 670 671 672 673 674 675 676 677 678 679 680 681
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665
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Fig. 2. Phylogenetic tree for the SmLgals3 gene. The phylogenetic tree was constructed based on the amino acid sequences of SmLgals3 from different species using the neighbor-joining method in MEGA 6. Gaps were removed by complete deletion and the phylogenetic tree was evaluated with 1,000 bootstrap replications. The bootstrapping values were indicated by numbers at the nodes. Dark solid circles indicated the newly characterized SmLgals3 genes.
EP
699 700 701 702 703 704 705 706 707 708 709 710 711 712 713
AC C
698
TE D
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682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697
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731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748
Fig. 3. Syntenic analysis of SmLgals3 genes from turbot, human, chicken, tetraoden, zebrafish, medaka, spotted gar, fugu. The Lgals3 gene is highlighted by bright blue color filled boxes. CDKN3: Cyclin-dependent kinase inhibitor 3; CNIH1: cornichon family AMPA receptor auxiliary protein 1; GMFB: Glia maturation factor beta; L2HGDH: L-2-hydroxyglutarate dehydrogenase; MED6: mediator complex subunit 6; SOS2: Ras/Rho guanine nucleotide exchange factor 2; ACTN1:actinin alpha 1.
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714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730
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Fig. 4. The tissue distribution of SmLgals3 in turbot. SmLgals3 expression in liver, skin, spleen, blood, head kidney, intestine, gill and brain was determined by quantitative real-time PCR. The expression level of SmLgals3 in skin was set as 1. The relative abundance of SmLgals3 was expressed as mean ± SE (N = 3).
765 766 767 768 769 770 771 772
AC C
EP
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760 761 762 763 764
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749 750 751 752 753 754 755 756 757 758 759
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Fig. 5. The expression patterns of SmLgals3 in turbot tissues at different timepoints (2h, 6h, 12h, and 24h) after V. anguillarum challenge. SmLgals3 expression in skin, intestine and gill was determined by quantitative real-time PCR. 18S rRNA was employed as an internal control. Asterisks (*) marked the significant differences between experimental and control groups (P < 0.05). Error bars indicated standard error (n = 3).
790 791 792 793 794 795 796 797
AC C
EP
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784 785 786 787 788 789
RI PT
773 774 775 776 777 778 779 780 781 782 783
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Fig. 6. The expression patterns of SmLgals3 in turbot mucosal tissues at different timepoints (2h, 4h, 8h, and 12h) after S. iniae challenge. SmLgals3 expression in skin, intestine and gill was determined by quantitative real-time PCR. 18S rRNA was employed as an internal control. Asterisks (*) marked the significant differences between experimental and control groups (P < 0.05). Error bars indicated standard error (n = 3).
AC C
EP
TE D
804 805 806 807 808 809 810 811 812 813 814 815
RI PT
798 799 800 801 802 803
816 817 818 819 820 821 822
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Fig. 7. Results of the in vitro binding assay of SmLgals3 on microbial ligands, including lipopolysaccharide, peptidoglycan, and lipoteichoic acid. * indicate a significant difference in the absorbance between different microbial ligands that exposed to r SmLgals3 and the control group: *p < 0.05; **p < 0.01.
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SC
823 824 825 826 827 828 829 830 831 832
834
AC C
EP
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833
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AC C
EP
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SC
835 836
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SmLgals3 gene is homologous to their counterparts in other vertebrates. SmLgals3 gene was ubiquitously expressed in turbot tissues.
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SmLgals3 had strong binding ability to LPS, LTA, and PGN.
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SmLgals3 gene showed different expression patterns following different bacterial challenge.