- Email: [email protected]
Molecular and functional characterization of a novel CD302 gene from ayu (Plecoglossus altivelis)
Accepted Manuscript Molecular and functional characterization of a novel CD302 gene from ayu (Plecoglossus altivelis) Shen-Xue Chen, Hai-Ling Ma, Yu-Hong Shi, Ming-Yun Li, Jiong Chen PII:
S1050-4648(16)30329-1
DOI:
10.1016/j.fsi.2016.05.022
Reference:
YFSIM 3979
To appear in:
Fish and Shellfish Immunology
Received Date: 17 March 2016 Revised Date:
27 April 2016
Accepted Date: 20 May 2016
Please cite this article as: Chen S-X, Ma H-L, Shi Y-H, Li M-Y, Chen J, Molecular and functional characterization of a novel CD302 gene from ayu (Plecoglossus altivelis), Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.05.022. 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.
ACCEPTED MANUSCRIPT 1
Molecular and functional characterization of a novel CD302 gene
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from ayu (Plecoglossus altivelis)
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Shen-Xue Chen1, Hai-Ling Ma1, Yu-Hong Shi1, Ming-Yun Li, Jiong Chen1, 2,*
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Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy
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Ningbo University, Ningbo 315211, China
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Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences,
Aquaculture, Ningbo University, Ningbo 315211, China
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*Corresponding author. Tel: +86 574 87609571; Fax: +86 574 87600167; E-mail
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address: [email protected] (J. Chen)
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ACCEPTED MANUSCRIPT Abstract
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Recognizing the presence of invading pathogens by pattern recognition receptors
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(PRRs) is key to mounting an effective innate immune response. Mammalian CD302
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is an unconventional C-type lectin like receptor (CTLR) involved in the functional
18
regulation of immune cells. However, the role of CD302 in fish remains unclear. In
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this study, we characterized a novel CD302 gene from ayu (Plecoglossus altivelis),
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which was tentatively named PaCD302. The cDNA sequence of PaCD302 is 1893
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nucleotides in length, and encodes a polypeptide of 241 amino acids with molecular
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weight 27.1 kDa and pI 4.69. Sequence comparison and phylogenetic tree analysis
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showed that PaCD302 is a type I transmembrane CTLR devoid of the known amino
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acid residues essential for Ca2+-dependent sugar binding. PaCD302 mRNA expression
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was detected in all tissues and cells tested, with the highest level in the liver.
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Following Vibrio anguillarum infection, PaCD302 mRNA expression was
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significantly upregulated in all tissues tested. For further functional analysis, we
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generated a recombinant protein for PaCD302 (rPaCD302) by prokaryotic expression
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and raised a specific antibody against rPaCD302. Western blot analysis revealed that
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the native PaCD302 is glycosylated. Refolded rPaCD302 was unable to bind to five
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monosaccharides (L-fucose, D-galactose, D-glucose, D-mannose and N-acetyl
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glucosamine) or two other polysaccharides (lipopolysaccharide and peptidoglycan). It
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was able to bind to three Gram-positive and seven Gram-negative bacteria, but show
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no bacterial agglutinating activity. PaCD302 function blocking using anti-PaCD302
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IgG resulted in inhibition of phagocytosis and bactericidal activity of ayu
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ACCEPTED MANUSCRIPT monocytes/macrophages (MO/MΦ), suggesting that PaCD302 regulates the function
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of ayu MO/MΦ. In summary, our study demonstrates that PaCD302 may participate
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in the immune response of ayu against bacterial infection via modulation of MO/MΦ
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function.
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Keywords:
Bactericidal
activity;
Bacterial
binding
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Monocyte/macrophage; Phagocytosis; Sugar binding capacity
capacity;
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CD302;
ACCEPTED MANUSCRIPT 1. Introduction
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The innate immune system, which is the first line of host defense, plays crucial roles
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in the early recognition of pathogens via cell-associated pattern recognition receptors
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(PRRs) [1]. These PRRs recognize distinct, conserved microbial structures called
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pathogen associated molecular patterns (PAMPs), including proteins, lipids,
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lipoproteins, glycans, and bacterial nucleic acids [2]. C-type lectin receptors (CLRs),
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one of the major PRRs, were originally defined by their carbohydrate-binding
51
function via a carbohydrate recognition domain (CRD). This domain enables them to
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bind complex saccharides displayed on various biological structures. However, a
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number of domains have been identified that are structurally homologous to the CRD,
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but do not always contain residues important for carbohydrate recognition and
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calcium coordination, and are not restricted to carbohydrate binding [3]. These
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domains have been termed C-type lectin-like domains (CTLD). Thus, the designation
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of C-type lectin-like receptors (CTLRs) was introduced to allow for the presence of
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one or more CTLD in the extracellular region. Many CTLRs have more than one
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function, but in general these relate to innate and adaptive immune cell activity, e.g.
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quick immune responses to potential pathogens. To date, a number of fish CTLRs
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have been identified and studied [4-10].
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CD302, also known as DCL-1, is a single-pass type I transmembrane CTLR with
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an N-terminal CTLD in the extracellular region [2,11]. The cDNA encoding CD302
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was first identified as a genetic fusion partner of human DEC-205 in Hodgkin’s
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lymphoma cell lines [12]. To date, studies on CD302 function have been limited to
ACCEPTED MANUSCRIPT mammals. The mRNA expression of human CD302 in leukocytes has been restricted
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to monocytes, macrophages, granulocytes, and dendritic cells, although its transcript
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was detected in many tissues [13]. Mammalian CD302 was considered an
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unconventional CTLR, which is not only involved in canonical endocytosis and
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phagocytosis [13], but may also be involved in cell adhesion and migration, and
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tumorigenesis [13-16]. In vitro experiments revealed that human CD302 had no
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binding capacity for four common sugars, namely mannnan, mannose, N-acetyl
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glucosamine (GlcNAc), and N-acetyl galactosamine (GalNAc) [13]. Given that all
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functional investigations to date have been focused on mammals, and there have been
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only limited sequence reports in lower vertebrates, studies regarding CD302 function
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in teleosts are certainly necessary. This will contribute towards a comprehensive
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understanding of the role of CD302 throughout evolution, as well as further
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delineating the teleost antimicrobial innate immune response [17-20].
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Ayu, Plecoglossus altivelis, the sole member of the Osmeriformes family
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Plecoglossidae, is an economically important fish found only in streams and coastal
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waters of Asia. In recent years, the ayu industry has grown rapidly in China, but
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bacterial diseases caused by Vibrio anguillarum have caused great loss to the ayu
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aquaculture [21]. Studies into the antibacterial immune response of ayu are therefore
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imperative. CTLRs play an important role in innate immunity and the innate immune
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system in teleosts is critical for protecting the organism against invading pathogens
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[5,7,10]. Therefore, understanding the function and mechanism of ayu CTLRs in
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shaping antimicrobial immunity may offer great potential for the future development
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of therapies for disease intervention. In the present study, we identified a novel
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CD302 homologue from ayu (PaCD302), and investigated its potential role in
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regulating monocytes/macrophages (MO/MΦ) function.
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2. Materials and methods
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2.1 Fish rearing
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Ayu were obtained from a commercial farm in Ninghai County, Ningbo city, China.
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Healthy fish, weighing 40–50 g each, were kept in 100-L tanks at 20–22°C. Fish were
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fed pelleted dry food once a day and acclimatized to laboratory conditions for two
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weeks prior to experimentation. Fish used in all investigations were healthy, with no
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history of pathological disease. All experiments were approved by the Committee on
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Animal Care and Use and the Committee on the Ethics of Animal Experiments of
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Ningbo University.
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2.2 Molecular characterization of PaCD302
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The cDNA sequence containing the complete open reading frame (ORF) of PaCD302
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was obtained from transcriptome data of ayu head kidney-derived MO/MΦ using a
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BLAST
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(http://www.cbs.dtu.dk/services/SignalP/) was used to predict the sequence of the
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signal peptide. SMART (http://smart.embl-heidelberg.de/) was used to predict the
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domain architecture of the putative protein. Potential N-glycosylation sites were
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predicted using the NetNGlyc1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/).
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Multiple
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search
sequence
(http://blast.ncbi.nlm.nih.gov/Blast.cgi).
alignment
was
analyzed
SignalP
using
4.1
ClustalW
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version 6.0 [22].
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2.3 Primary culture of ayu head kidney-derived MO/MΦ
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Ayu head kidney-derived MO/MΦ were isolated as previously described [23], with
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some modifications. Briefly, head kidneys were aseptically extracted, and dissociated
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in RPMI 1640 (Invitrogen, Shanghai, China) supplemented with 2% fetal bovine
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serum (FBS) (Invitrogen), penicillin (100 U/ml), streptomycin (100 mg/ml), and
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heparin (20 U/ml). Cells were separated using Ficoll-Hypaque PREMIUM (1.077
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g/ml) (GE Healthcare, New Jersey, USA) in combination with centrifugation
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according to the manufacturer's instructions. Cells were then seeded onto 35-mm well
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plates at a density of 2 × 106 cells per well and allowed to adhere overnight at 24°C in
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an incubator with 5% CO2. The medium was changed to complete medium (4% ayu
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serum, 6% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin), and cells were kept in
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the incubator under the same conditions.
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2.4 Sample preparation for PaCD302 mRNA expression analysis
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Ayu tissues including liver, muscle, head kidney, brain, spleen, intestine, gill, MO/MΦ,
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peripheral blood lymphocytes (PBLs) were collected from healthy fish for tissue
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PaCD302 mRNA expression profile analysis. For pathologically correlated expression
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analysis, fish were challenged by intraperitoneal injection with 1.2x104 colony
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forming units (CFUs) live V. anguillarum (in 100 µl PBS) per fish, and PBS alone was
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used as a control. Liver, head kidney, spleen, and PBLs samples were collected at 4, 8,
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12, and 24 h post infection (hpi). Head kidney-derived MO/MΦ were purified as
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ACCEPTED MANUSCRIPT described in Section 2.3 and infected with live V. anguillarum at a multiplicity of
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infection (MOI) of 10. Tissues were immediately snap-frozen in liquid nitrogen and
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preserved in -80°C until subsequent use. PBLs and MO/MΦ were resuspended in
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RNAiso (TaKaRa, Dalian, China) before RNA extraction.
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2.5 Real-time quantitative PCR (qPCR) assay
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Total RNA was extracted using RNAiso reagents (TaKaRa), followed by
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deoxyribonuclease I digestion to eliminate genomic DNA. Specific primers for
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PaCD302
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AGCAAGAAGGTGTGGAAGGAT-3′
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GAGGTCAGAAGAGAAGCCAC-3′, which was expected to amplify a 135 base pair
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(bp)
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5′-TCGTGCGTGACATCAAGGAG-3′
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5′-CGCACTTCATGATGCTGTTG-3′, was used to amplify a 231-bp fragment from
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β-actin, which is widely used as an internal control in ayu [8,19]. The qPCR reaction
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was performed in an RT-Cycler™ Realtime Fluorescence Quantitative PCR
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thermocycler (CapitalBio, Beijing, China) using SYBR premix Ex Taq (Perfect Real
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Time) (TaKaRa). The reaction mixture was incubated for 5 min at 95°C, followed by
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40 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. The mRNA expression of
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PaCD302 was normalized to that of β-actin using the 2-∆∆Ct method [24].
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2.6 Preparation of recombinant PaCD302 (rPaCD302)
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The primers rPaCD302 (+): 5′-GGAATTCGATTGCCCTGCGGATGGG-3′ and
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rPaCD302 (-): 5′-GCTCGAGCTGGCACACCACCCCATTC-3′ (underlined are the
Another
PaCD302-T
and
PaCD302-T
primer
pair,
(+):
5′-
(−):
5′-
pActin2
and
(+):
pActin2(−):
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were
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ACCEPTED MANUSCRIPT EcoRI and XhoI sites, respectively) were used to amplify the sequence encoding the
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extracellular region of PaCD302. Amplicons of the expected size were digested using
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EcoRI and XhoI and cloned into the pET28a(+) vector. The recombinant plasmid
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pET28a-PaCD302 was transformed into Escherichia coli BL21 (DE3). rPaCD302
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was overexpressed as inclusion bodies, which were dissolved in an 8 M urea solution
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and purified using a nickel-nitrilotriacetic acid (Ni-NTA) column (QIAGEN, Hilden,
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Germany) at 4°C. Refolding of solubilized rPaCD302 using 8 to 2 M urea gradient
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size-exclusion chromatography was performed on an XK 16/100 column packed with
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Superdex 75 gel media (GE Healthcare) at 4°C. Peak fractions were pooled and
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concentrated using a 10,000 NMWL spin filter (Millipore, Shanghai, China) at 4°C.
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The eluted fractions were desalted on a Bio-Gel P-6 desalting column (Bio-Rad,
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Shanghai, China), then lyophilized and stored at −80°C before use.
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2.7 Antibody preparation and western blot assay
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Antibody production was performed as previously reported [8]. Briefly, rPaCD302
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emulsified with Freund’s incomplete adjuvant was used to immunize mice by
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intraperitoneal injection once every seven days for a total of four injections. Control
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mice were injected with complete Freund’s adjuvant. Whole blood was collected and
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centrifuged to obtain sera. Anti-rPaCD302 IgG and mouse isotype IgG were purified
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using protein G chromatography media (Bio-Rad).
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Western blot was performed to detect native PaCD302 in ayu MO/MΦ using the
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prepared antibody. Briefly, total protein was extracted from MO/MΦ, resolved by
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SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Mouse
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anti-PaCD302 antiserum was used as the primary antibody at a 1:1000 dilution,
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followed by horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (1:5000) as
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the
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chemiluminescence (ECL) kit (Advansta, Menlo Park, USA). In order to determine
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whether native PaCD302 is N-glycosylated, denatured proteins of ayu MO/MΦ were
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treated with PNGase F (New England Biolabs, Beverly, USA) at 37°C overnight, and
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analyzed by western blot.
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2.8 Sugar binding assay
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An indirect enzyme-linked immunosorbent assay (ELISA) was performed on high
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affinity ELISA plates (Corning Costar, NY, USA) to detect the binding capacity of
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rPaCD302 to five monosaccharides, L-fucose, D-galactose, D-glucose, D-mannose
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and GlcNAc, and two polysaccharides, lipopolysaccharide (LPS) (E. coli 055: B5)
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and peptidoglycan (PGN) (Staphylococcus aureus) (Sigma Chemical Co., St. Louis,
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USA), according to previously described methods [8,25]. Briefly, each well of a
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microplate was coated with 50 µl of sugar (80 µg/ml) in TBS (10 mM Tris-HCl,
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pH7.5, 150 mM NaCl) and incubated overnight at 37°C. After heating at 60°C for 30
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min, the plate was blocked with 50 µl of 1 mg/ml BSA per well for 2 h at 37°C, and
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washed four times with TBS. rPaCD302 (0–100 µg/ml protein, diluted with TBS
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containing 0.1 mg/ml of BSA) was added to each well and incubated for 3 h at 30°C.
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After washing with TBS to remove free rPaCD302, 100 µl of anti-PaCD302 IgG
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(1:2000 diluted) was added to each well and incubated for 1 h at 37°C. After washing
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with TBS to remove free anti-PaCD302 IgG, 100 µl of peroxidase-conjugated goat
were
visualized
using
an
enhanced
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Stabilized 3, 3′, 5, 5′-tetramethylbenzidine (Sigma Chemical Co.) solution containing
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hydrogen peroxide was added to each well, the plate was incubated at room
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temperature and the absorbance of each well was measured using a SpectraMax® M3
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multi-mode microplate reader (Molecular Devices, Sunnyvale, USA). The negative
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control used TBS instead of rPaCD302. All experiments were repeated three times.
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2.9 Bacterial binding and agglutination assay of rPaCD302
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Three Gram-positive bacteria (Listeria monocytogenes, S. aureus, and Streptococcus
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iniae) and seven Gram-negative bacteria (Aeromonas hydrophila, E. coli,
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Edwardsiella tarda, Vibrio alginolyticus, V. anguillarum, Vibrio harveyi, and Vibrio
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parahaemolyticus) were selected for the assay. Bacteria were cultured overnight to
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analyze the binding capacity of rPaCD302 as previously described [25] in the
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presence or absence of 10 mM CaCl2. After centrifugation at 6000 × g for 5 min, cell
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pellets were collected, washed twice with TBS, then thoroughly resuspended in TBS.
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Resuspended bacteria (500 µl, 2 × 108 cells/ml) were incubated with rPaCD302 (final
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concentration 0.5 mg/ml) at room temperature for 30 min. Microorganisms were
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pelleted, washed four times with TBS, and eluted with 7% SDS for 1 min. Cells
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incubated with TBS only were used as a control. Pellets were subjected to SDS-PAGE,
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and the presence of rPaCD302 was determined using western blot.
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Bacterial agglutination activity of rPaCD302 was assayed as previously described
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[8]. Cells were harvested at the mid-logarithmic phase, and were resuspended to 2 ×
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108 cells per ml in TBS. The microorganism/TBS solution (25 µl) was incubated with
ACCEPTED MANUSCRIPT 25 µl rPaCD302/TBS (200 µg/ml) at 28ºC for 1 h in the presence or absence of 10
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mM CaCl2. Agglutination of bacteria was analyzed by a bright-field microscopy
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OLYMPUS BH-2 (Olympus, Tokyo, Japan).
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2.10 In vitro phagocytosis assay of ayu MO/MΦ
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Logarithmic phase E. coli DH5α were labeled with fluorescein isothiocyanate (FITC)
225
(Sigma Chemical Co.) according to the manufacturer’s protocol, and FITC-labeled
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bacteria were hereafter designated as E. coli-FITC. For neutralization analysis, ayu
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MO/MΦ grown on cover slips in 35 mm plates (2 × 106 cells/well) were pre-incubated
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with 200 µg/ml anti-PaCD302 IgG or isotype IgG for 30 min. PBS was used as a
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control. E. coli-FITC was added at a MOI of 10, and cells were further incubated for
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30 min. Trypan blue (0.4%) was used to quench the fluorescence that resulted from
231
particles that were outside of the cells or sticking to the cell surface. Engulfed bacteria
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were examined using a Gallios flow cytometer (Beckman Coulter, Miami, USA) and
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data were analyzed using FlowJo software. Relative mean fluorescence intensity (MFI)
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of the IgG-treated group was expressed as fold-change relative to the value of the
235
no-bacteria control, and the value of the isotype IgG-treated group was assigned a unit
236
of 100.
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2.11 Assay of MO/MΦ bactericidal activity
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Ayu MO/MΦ were pre-incubated with 200 µg/ml anti-PaCD302 IgG or isotype IgG
239
for 30 min, and then infected with live V. anguillarum at a MOI of 10. MO/MΦ
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phagocytosis of bacteria was allowed to proceed for 30 min at 24°C in an atmosphere
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of 5% CO2, and noninternalized V. anguillarum were removed by washing with sterile
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ACCEPTED MANUSCRIPT PBS. One set of samples (the uptake group) was lysed in a 1% Triton X-100 solution
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and plated onto solid thiosulfate citrate bile salts sucrose (TCBS) agar medium to
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provide bacterial uptake values. The remaining set (the kill group) was incubated for a
245
further 1.5 h to allow bacterial killing to occur before cell lysis. After incubation at
246
30°C for 18 h, the number of colonies on each plate was counted. Bacterial survival
247
rate was determined by dividing the CFU of the kill group by that of the uptake group.
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Three independent experiments were carried out.
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2.12 Statistical analysis
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All data were reported as mean ± SEM. Statistical analysis of the results was
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conducted using one-way Analysis of Variance (ANOVA) with SPSS version 13.0
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(SPSS Inc, Chicago, USA). P values less than 0.05 were considered statistically
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significant.
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3. Results
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3.1 Cloning and sequence analysis of PaCD302
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The cDNA sequence of PaCD302 was identified by a BLAST search and submitted to
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the DDBJ/EMBL/GenBank databases under accession number JP773538. The cDNA
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is 1893 nucleotides (nt) in length and contains a large open reading frame (ORF) of
260
726 nt, which is predicted to encode a 241 amino acid (aa) polypeptide with a
261
calculated molecular weight (MW) of 27.1 kDa and a pI of 4.69. PaCD302 is the
262
simplest type I transmembrane C-type lectin discovered to date; it contains a signal
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peptide (aa 1-26), one CTLD (aa 29-163), and one short spacer followed by one
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ACCEPTED MANUSCRIPT transmembrane domain (aa 179-201) and one cytoplasmic tail (Fig. 1A). The
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asparagine (N) residue at aa position 149 is predicted to be an N-glycosylated site (Fig.
266
1A). The PaCD302 CTLD contains 6 cysteine (C) residues, which is a typical feature
267
of a C-type lectin motif, forming an intramolecular disulfide bond-mediated C-type
268
lectin fold (Fig. 1A). Multiple alignment shows that PaCD302 CTLD is devoid of the
269
known amino acid residues essential for Ca2+-dependent sugar binding, i.e. the EPN
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motif (for mannose binding), the QPD motif (for galactose binding), and the WND
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motif (for Ca2+ binding) (Fig. 1B).
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Fig. 1. Multiple alignment of the amino acid sequences of PaCD302 and related
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proteins from other vertebrates. (A) Comparison of amino acid sequences of
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vertebrate CD302. Conserved cysteine residues are marked as “*”. The asparagine
276
residue, predicted to be an N-glycosylated residue, is marked as “
277
of CTLD sequences from human dendritic cell-specific intercellular adhesion
”. (B) Comparison
ACCEPTED MANUSCRIPT molecule-3-grabbing non-integrin (hDC-SIGN), human macrophage galactose
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binding lectin (hMGL), ayu C-type lectin receptor C (PaCTLRC) and PaCD302. The
280
single and double underlines indicate the positions of the EP(N/Q)PD and WND
281
motifs, respectively. Threshold for shading was 70%; similar residues are marked with
282
a gray shadow, identical residues with a black shadow, and alignment gaps with “-”.
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Accession numbers: ayu CD302, JP773538; black rockcod (Notothenia coriiceps)
284
CD302, XM_010782533; rainbow trout (Oncorhynchus mykiss) CD302, FR904281;
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Japanese ricefish (Oryzias latipes) CD302, XM_004081729; large yellow croaker
286
(Larimichthys crocea) CD302, KQ042842; Nile tilapia (Oreochromis niloticus)
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CD302, XM_005452988; zebrafish (Danio rerio) CD302, XM_002667634; human
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(Homo sapiens) CD302, NM_014880; PaCTLRC, KP329196; hDC-SIGN, AF290886;
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hMGL, BC039011.
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Sequence comparison showed that PaCD302 shared the highest aa identity (65%)
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with CD302 from black rockcod. Phylogenetic tree analysis of amino acid sequences
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showed that PaCD302 falls into the group of fish CD302, but is distant from other
293
smaller clusters (Fig. 2).
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Fig. 2. Phylogenetic analysis of amino acid sequences of CD302 using the
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neighbor-joining method. Human CD302 was used as an outgroup to root the tree.
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Values at the forks indicate the percentage of trees in which this grouping occurred
298
after bootstrapping (1,000 replicates; shown only when > 60%). Scale bar shows
299
number of substitutions per base. Accession numbers are listed in the legend of Fig. 1
300
and below: Mexican tetra (Astyanax mexicanus), XM_007230421; Atlantic salmon
301
(Salmo
302
XM_008328697; zebra mbuna (Maylandia zebra), XM_004557639; killfish
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(Austrofundulus limnaeus), XM_014016681; Amazon molly (Poecilia formosa),
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XM_007546599; spotted green pufferfish (Tetraodon nigroviridis), CAAE01015004;
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tiger pufferfish (Takifugu rubripes), XM_003961965; northern pike (Esox lucius),
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XM_010890847; southern platyfish (Xiphophorus maculatus), XM_005805321.
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3.2 qPCR analysis of PaCD302 mRNA expression
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qPCR was used to analyze the tissue mRNA expression profile of PaCD302 in healthy
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XM_014164142;
tongue
sole
(Cynoglossus
semilaevis),
AC C
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ACCEPTED MANUSCRIPT ayu. PaCD302 mRNA was constitutively expressed in all tissues tested, including
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liver, heart, brain, spleen, gill, intestine, muscle, head kidney, PBLs, and MO/MΦ,
311
with the highest expression in the liver (Fig. 3A). Upon V. anguillarum infection,
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PaCD302 mRNA expression was found to be upregulated (Fig. 3B-F). PaCD302
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mRNA expression in the liver, spleen and PBLs was upregulated at 4 hpi, followed
314
thereafter by a decrease. The highest transcript levels of PaCD302 in the head kidney
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occurred at 24 hpi (Fig. 3B-E). The most significant increase in PaCD302 transcript
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levels was observed in the liver at 4 hpi (16.8-fold) (Fig. 3B-E).
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Fig. 3. qPCR analysis of PaCD302 transcripts in ayu tissues and cells. (A) mRNA
ACCEPTED MANUSCRIPT expression profiles of PaCD302 in healthy ayu tissues and cells. L: liver, H: heart, B:
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brain, S: spleen, MO/MΦ: monocytes/macrophages, G: gill, I: intestine, M: muscle,
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HK: head kidney, PBLs: peripheral blood lymphocytes. (B-F) Changes in PaCD302
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mRNA expression in tissues and cells of the ayu immune system, following V.
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anguillarum infection. Tissues and cells were collected at different time points
324
post-bacterial infection. PaCD302 mRNA expression was normalized to β-actin. Data
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are expressed as mean ± SEM of results from four individual fish. * P < 0.05.
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3.3 Prokaryotic expression, purification and antibody preparation of rPaCD302
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The recombinant protein was successfully expressed in E. coli with a MW of 24.5
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kDa in SDS-PAGE, which was 4.1 kDa larger than the MW calculated from sequence
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data (20.4 kDa, comprising 15.7 kDa protein and 4.7 kDa His-tag) (Fig. 4A). The
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aberrant SDS–PAGE behavior of PaCD302 may be due to mobility retardation of the
331
His-tag [26] and the reduced protein structure [12]. The overexpressed protein was
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extracted from inclusion bodies, purified by affinity chromatography using an
333
Ni-NTA column, and refolded by 8 to 2 M urea gradient size-exclusion
334
chromatography (Fig. 4A). rPaCD302 was then used to immunize mice to produce the
335
antiserum. Using this antiserum, we determined that the MW of the native PaCD302
336
in MO/MΦ was about 32 kDa by western blot analysis (Fig. 4B), which was bigger
337
than that calculated from sequence data (27.1 kDa). After PNGase F digestion, the
338
MW of the native PaCD302 decreased to about 27 kDa, suggesting the existence of
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post-translational N-glycosylation, as was previously reported for human CD302 [13].
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Anti-rPaCD302 IgG was purified from antisera using protein G chromatography
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media for subsequent use.
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Fig. 4. Prokaryotic expression of PaCD302 and western blot analysis. (A) 12%
345
SDS–PAGE analysis of bacterial lysates and purified rPaCD302. Lane M: protein
346
marker; Lane 1: lysates of pET28a-PaCD302 transformed E. coli BL21 without IPTG
347
induction; Lane 2: lysates of pET28a-PaCD302 transformed E. coli BL21 with IPTG
348
induction; Lane 3: purified rPaCD302. (B) Western blot analysis of PaCD302 using
349
antisera against rPaCD302. Lane 4: total protein extracted from ayu MO/MΦ; Lane 5:
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total protein extracted from ayu MO/MΦ, PNGase F digested; Lane 6: E. coli BL21
351
lysates, negative control.
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3.4 Carbohydrate and bacteria binding capacity of rPaCD302
353
We investigated the OD 450 values, which reflect the binding capacity of rPaCD302,
354
using a carbohydrate binding assay. rPaCD302 was unable to bind five
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ACCEPTED MANUSCRIPT monosaccharides and two polysaccharides. Western blot analysis was used to
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determine the bacteria binding capacity, which revealed that rPaCD302 was able to
357
bind all ten of the bacteria tested, in the presence of calcium ions. Binding of
358
rPaCD302 to S. iniae and E. tarda was relatively weaker than it was to other bacteria
359
(Fig. 5). However, rPaCD302 did not agglutinate all microorganisms tested (data not
360
shown).
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Fig. 5. Western blot analysis of bacteria binding activity of rPaCD302 in the presence
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(A) or absence (B) of Ca2+ by using a specific antiserum to PaCD302. PC: rPaCD302
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used as a positive control, Lmo: L. monocytogenes, Sau: S. aureus, Sin: S. iniae, Ahy:
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A. hydrophila, Eco: E. coli, Eta: E. tarda, Val: V. alginolyticus, Van: V. anguillarum,
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Vha: V. harveyi, Vpa: V. parahaemolyticus.
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3.5 PaCD302 effect on phagocytosis and bacterial killing by ayu MO/MΦ
368
Human PaCD302 has been reported to be involved in phagocytosis by human MΦ
369
[13]. We therefore used the anti-PaCD302 IgG to block PaCD302 function in order to
370
investigate whether it has an effect on phagocytosis and bacterial killing by ayu
371
MO/MΦ. Compared to the isotype IgG group, the phagocytic activity of E. coli-FITC
372
by anti-PaCD302 IgG-treated MO/MΦ decreased by 28.3% (Fig. 6A), while the
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ACCEPTED MANUSCRIPT survival rate of V. anguillarum increased from 54.49 ± 4.82% to 75.2 ± 5.49% (Fig.
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6B).
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Fig. 6. Effect of PaCD302 blockage on phagocytosis and bacterial killing by ayu
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MO/MΦ. Anti-PaCD302 IgG was used to block PaCD302 function on MO/MΦ,
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while isotype IgG was used as a control. (A) Flow cytometry analysis of E. coli-FITC
379
phagocytosis by ayu MO/MΦ. Relative mean fluorescence intensity (MFI) of
380
anti-PaCD302 IgG- or isotype IgG-treated group expressed as fold-change, and the
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value of isotype control was assigned a unit of 100. Data are mean ± SD of three
382
repeats. *P < 0.05. (B) Killing of V. anguillarum by ayu MO/MΦ was measured using
383
a CFU assay. Data are expressed as mean ± SD of three repeats. *P < 0.05.
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4. Discussion
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CTLRs comprise a heterogeneous group of transmembrane proteins that are able to
387
recognize pathogens and in some cases engage signaling pathways that propagate into
388
cells to elicit microbicidal or inflammatory responses [8,20,27]. In the present study,
389
we identified for the first time a CD302 homologue in ayu. PaCD302 is predicted to
390
be a type I transmembrane CTLR with a signal peptide, a CTLD, a short spacer
391
followed by a transmembrane domain and a cytoplasmic domain. PaCD302 mRNA is
392
widely expressed in ayu tissues and immune cells, and is especially abundant in the
393
liver, which is similar to that reported for other fish CTLRs [7,9,28]. CD302 in
394
mammalian leukocytes has been restricted to monocytes, macrophages, granulocytes,
395
and dendritic cells. Liver contains blood and many immune cells such as
396
tissue-resident mature macrophages-Kupffer cells. On the other hand, head
397
kidney-derived MO/MΦ is in an immature state. Therefore, the mRNA expression
398
levels of PaCD302 in several tissues, especially the liver, were higher than that in
399
MO/MΦ. The transcript of PaCD302 in MO/MΦ was higher than that in PBLs is
400
possibly caused by the percentage of cells expressing CD302. When ayu was infected
401
by V. anguillarum, PaCD302 mRNA expression was upregulated in all tissues tested
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and in immune cells. These results suggest that PaCD302 should be involved in the
403
innate immune response of ayu against bacterial infection.
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Unlike Ca2+-dependent CRDs found in prototypical CTLRs such as
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DC-SIGN/CD209, PaCD302 CTLD is devoid of the amino acid residues known to be
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407
have classic sugar binding capacity. In a previous report, human CD302 was shown to
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be unable to bind mannan, mannose, GlcNAc, and GalNAc [13]. In the present study,
409
we confirmed that PaCD302 was not able to bind all tested saccharides, which is in
410
agreement with that predicted from sequence analysis and that reported for human
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CD302. A number of CTLRs exist today for which only a few, and sometimes no,
412
carbohydrate ligands have been identified [2]. The nonclassical CTLRs have evolved
413
to bind sugar and nonsugar ligands without classic Ca2+ and sugar binding motifs, for
414
example, dectin-1 for β-glucan in a Ca2+-independent manner [29]. It is possible that
415
PaCD302 has some alternative carbohydrate binding capacity like dectin-1. It is also
416
possible that PaCD302 binds to protein ligands in the same manner that CD209a
417
binds to LECT2 [30] and that P2X7R binds to cathelicidin [18]. It will be interesting
418
to determine whether PaCD302 has a binding capacity for alternative carbohydrates or
419
other biological structures in the future.
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Agglutinating activity of lectins is a basic reaction against invading pathogens,
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and binding is the first step in this process [31]. In the present study, we tested the
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bacteria agglutination activity of rPaCD302 and showed that it was able to bind all ten
423
bacteria tested, but no obvious agglutination was observed. In addition to bacterial
424
saccharides like LPS and PGN, other PAMPs such as lipoproteins, flagellin, bacterial
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nucleic acid structures are also the possible ligands of CTLRs. For examples,
426
DEC-205 could bind to plasminogen activator (PLA) on the surface of Yersinia pestis,
427
which mediates bacterial attachment [32]. DC-SIGN could bind to Surface (S) layer A
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ACCEPTED MANUSCRIPT protein (SlpA) on the surface of Lactobacillus acidophilus [33]. In this study,
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PaCD302 did not bind to classical saccharides, but it did bind to many bacteria. If
430
there are bacterial PAMPs recognized by PaCD302, they are likely to have a high pI
431
and be rich in basic amino acids because the PaCD302 extracellular domain is rich in
432
acidic amino acids. There have been reports of other C-type lectins that can only bind
433
bacteria but show no bacterial agglutinating activity. For example, Pc-Lec1 and
434
Pc-Lec2 from red swamp crayfish (Procambarus clarkia) did not agglutinate any of
435
the Gram-positive and Gram-negative bacteria tested, but they were able to bind to
436
them [34,35]. Thus, our result suggests that PaCD302 may serve as a “sensor,” which
437
can recognize invading pathogens, bind to them, and then initiate an immune response,
438
rather than agglutinating them.
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At present, it is known that fish possess a well-developed non-specific immune
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system and that phagocytosis plays an important role in their defense against
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microorganisms [36]. Due to the importance of this process, several studies have
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focused on CTLRs that mediate phagocytosis in fish. For example, mannose receptor
443
(MR) from blunt bream carp (Megalobrama amblycephala) is involved in the
444
macrophage engulfment of GFP-expressing E. coli, resulting in a respiratory burst,
445
nitric oxide production and inflammatory cytokine secretion [37]. In our previous
446
reports, another two CTLRs from ayu, PaCD209L and PaCTLRC, were shown to
447
recognize pathogens and participate in phagocytosis and the bacterial killing process
448
of ayu MO/MΦ [8,25]. In the present study, we observed that function-blocking of
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PaCD302 effectively inhibited phagocytosis and bacterial killing of ayu MO/MΦ,
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ACCEPTED MANUSCRIPT revealing that PaCD302 should also function as a PRR. A previous report on human
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CD302 showed that the effect of function-blocking on macrophage phagocytosis was
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about 8-fold less efficient than the effect of macrophage MR or DEC-205
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function-blocking on macrophage phagocytosis [13]. Our study also revealed that the
454
effect of PaCD302 function-blocking on MO/MΦ phagocytosis of E. coli-FITC
455
(approximately 71.7% of the control) was obviously less efficient than the effect of
456
PaCD209L function-blocking (approximately 25.1% of the control) [25]. This is in
457
agreement with findings from mammals [13], and means that CD302 could also have
458
a weaker influence than other CTLRs on macrophage phagocytosis in fish. Human
459
CD302 was reported to colocalize with F-actin, suggesting it may play a role in cell
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adhesion and migration [13]. However, this remains to be confirmed in fish.
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In conclusion, we have identified the characteristics of a novel CD302 in ayu,
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revealing it to be an unconventional CTLR. PaCD302 mRNA was significantly
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upregulated upon bacterial challenge in all tissues tested. PaCD302 participated in the
464
phagocytosis and bacterial killing processes of ayu MO/MΦ. This is the first report on
465
the functional analysis of a fish CD302-like protein, and our data demonstrate that
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PaCD302 may play a role in the ayu immune response to bacterial infection.
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Acknowledgments
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This project was supported by the Program for the National Natural Science
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Foundation of China (31372555), the Research Project of Chinese Ministry of
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Education (213017A), Zhejiang Provincial Natural Science Foundation of China
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(LZ13C190001), and the KC Wong Magna Fund in Ningbo University.
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ACCEPTED MANUSCRIPT Highlights
> We characterized a novel CD302 (PaCD302) gene from ayu.
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> PaCD302 mRNA expression responded to Vibrio anguillarum infection. > PaCD302 did not bind seven common saccharides such as d-mannose, LPS and PGN.
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> PaCD302 bound to ten microorganisms, but did not agglutinate them.
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> PaCD302 regulated the phagocytic and bactericidal activities of MO/MΦ in vitro.
S1050-4648(16)30329-1
DOI:
10.1016/j.fsi.2016.05.022
Reference:
YFSIM 3979
To appear in:
Fish and Shellfish Immunology
Received Date: 17 March 2016 Revised Date:
27 April 2016
Accepted Date: 20 May 2016
Please cite this article as: Chen S-X, Ma H-L, Shi Y-H, Li M-Y, Chen J, Molecular and functional characterization of a novel CD302 gene from ayu (Plecoglossus altivelis), Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.05.022. 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.
ACCEPTED MANUSCRIPT 1
Molecular and functional characterization of a novel CD302 gene
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from ayu (Plecoglossus altivelis)
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Shen-Xue Chen1, Hai-Ling Ma1, Yu-Hong Shi1, Ming-Yun Li, Jiong Chen1, 2,*
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Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy
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Ningbo University, Ningbo 315211, China
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Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences,
Aquaculture, Ningbo University, Ningbo 315211, China
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*Corresponding author. Tel: +86 574 87609571; Fax: +86 574 87600167; E-mail
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address: [email protected] (J. Chen)
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ACCEPTED MANUSCRIPT Abstract
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Recognizing the presence of invading pathogens by pattern recognition receptors
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(PRRs) is key to mounting an effective innate immune response. Mammalian CD302
17
is an unconventional C-type lectin like receptor (CTLR) involved in the functional
18
regulation of immune cells. However, the role of CD302 in fish remains unclear. In
19
this study, we characterized a novel CD302 gene from ayu (Plecoglossus altivelis),
20
which was tentatively named PaCD302. The cDNA sequence of PaCD302 is 1893
21
nucleotides in length, and encodes a polypeptide of 241 amino acids with molecular
22
weight 27.1 kDa and pI 4.69. Sequence comparison and phylogenetic tree analysis
23
showed that PaCD302 is a type I transmembrane CTLR devoid of the known amino
24
acid residues essential for Ca2+-dependent sugar binding. PaCD302 mRNA expression
25
was detected in all tissues and cells tested, with the highest level in the liver.
26
Following Vibrio anguillarum infection, PaCD302 mRNA expression was
27
significantly upregulated in all tissues tested. For further functional analysis, we
28
generated a recombinant protein for PaCD302 (rPaCD302) by prokaryotic expression
29
and raised a specific antibody against rPaCD302. Western blot analysis revealed that
30
the native PaCD302 is glycosylated. Refolded rPaCD302 was unable to bind to five
31
monosaccharides (L-fucose, D-galactose, D-glucose, D-mannose and N-acetyl
32
glucosamine) or two other polysaccharides (lipopolysaccharide and peptidoglycan). It
33
was able to bind to three Gram-positive and seven Gram-negative bacteria, but show
34
no bacterial agglutinating activity. PaCD302 function blocking using anti-PaCD302
35
IgG resulted in inhibition of phagocytosis and bactericidal activity of ayu
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ACCEPTED MANUSCRIPT monocytes/macrophages (MO/MΦ), suggesting that PaCD302 regulates the function
37
of ayu MO/MΦ. In summary, our study demonstrates that PaCD302 may participate
38
in the immune response of ayu against bacterial infection via modulation of MO/MΦ
39
function.
40
Keywords:
Bactericidal
activity;
Bacterial
binding
42
Monocyte/macrophage; Phagocytosis; Sugar binding capacity
capacity;
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CD302;
ACCEPTED MANUSCRIPT 1. Introduction
45
The innate immune system, which is the first line of host defense, plays crucial roles
46
in the early recognition of pathogens via cell-associated pattern recognition receptors
47
(PRRs) [1]. These PRRs recognize distinct, conserved microbial structures called
48
pathogen associated molecular patterns (PAMPs), including proteins, lipids,
49
lipoproteins, glycans, and bacterial nucleic acids [2]. C-type lectin receptors (CLRs),
50
one of the major PRRs, were originally defined by their carbohydrate-binding
51
function via a carbohydrate recognition domain (CRD). This domain enables them to
52
bind complex saccharides displayed on various biological structures. However, a
53
number of domains have been identified that are structurally homologous to the CRD,
54
but do not always contain residues important for carbohydrate recognition and
55
calcium coordination, and are not restricted to carbohydrate binding [3]. These
56
domains have been termed C-type lectin-like domains (CTLD). Thus, the designation
57
of C-type lectin-like receptors (CTLRs) was introduced to allow for the presence of
58
one or more CTLD in the extracellular region. Many CTLRs have more than one
59
function, but in general these relate to innate and adaptive immune cell activity, e.g.
60
quick immune responses to potential pathogens. To date, a number of fish CTLRs
61
have been identified and studied [4-10].
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CD302, also known as DCL-1, is a single-pass type I transmembrane CTLR with
63
an N-terminal CTLD in the extracellular region [2,11]. The cDNA encoding CD302
64
was first identified as a genetic fusion partner of human DEC-205 in Hodgkin’s
65
lymphoma cell lines [12]. To date, studies on CD302 function have been limited to
ACCEPTED MANUSCRIPT mammals. The mRNA expression of human CD302 in leukocytes has been restricted
67
to monocytes, macrophages, granulocytes, and dendritic cells, although its transcript
68
was detected in many tissues [13]. Mammalian CD302 was considered an
69
unconventional CTLR, which is not only involved in canonical endocytosis and
70
phagocytosis [13], but may also be involved in cell adhesion and migration, and
71
tumorigenesis [13-16]. In vitro experiments revealed that human CD302 had no
72
binding capacity for four common sugars, namely mannnan, mannose, N-acetyl
73
glucosamine (GlcNAc), and N-acetyl galactosamine (GalNAc) [13]. Given that all
74
functional investigations to date have been focused on mammals, and there have been
75
only limited sequence reports in lower vertebrates, studies regarding CD302 function
76
in teleosts are certainly necessary. This will contribute towards a comprehensive
77
understanding of the role of CD302 throughout evolution, as well as further
78
delineating the teleost antimicrobial innate immune response [17-20].
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Ayu, Plecoglossus altivelis, the sole member of the Osmeriformes family
80
Plecoglossidae, is an economically important fish found only in streams and coastal
81
waters of Asia. In recent years, the ayu industry has grown rapidly in China, but
82
bacterial diseases caused by Vibrio anguillarum have caused great loss to the ayu
83
aquaculture [21]. Studies into the antibacterial immune response of ayu are therefore
84
imperative. CTLRs play an important role in innate immunity and the innate immune
85
system in teleosts is critical for protecting the organism against invading pathogens
86
[5,7,10]. Therefore, understanding the function and mechanism of ayu CTLRs in
87
shaping antimicrobial immunity may offer great potential for the future development
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of therapies for disease intervention. In the present study, we identified a novel
89
CD302 homologue from ayu (PaCD302), and investigated its potential role in
90
regulating monocytes/macrophages (MO/MΦ) function.
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2. Materials and methods
93
2.1 Fish rearing
94
Ayu were obtained from a commercial farm in Ninghai County, Ningbo city, China.
95
Healthy fish, weighing 40–50 g each, were kept in 100-L tanks at 20–22°C. Fish were
96
fed pelleted dry food once a day and acclimatized to laboratory conditions for two
97
weeks prior to experimentation. Fish used in all investigations were healthy, with no
98
history of pathological disease. All experiments were approved by the Committee on
99
Animal Care and Use and the Committee on the Ethics of Animal Experiments of
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Ningbo University.
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2.2 Molecular characterization of PaCD302
102
The cDNA sequence containing the complete open reading frame (ORF) of PaCD302
103
was obtained from transcriptome data of ayu head kidney-derived MO/MΦ using a
104
BLAST
105
(http://www.cbs.dtu.dk/services/SignalP/) was used to predict the sequence of the
106
signal peptide. SMART (http://smart.embl-heidelberg.de/) was used to predict the
107
domain architecture of the putative protein. Potential N-glycosylation sites were
108
predicted using the NetNGlyc1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/).
109
Multiple
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search
sequence
(http://blast.ncbi.nlm.nih.gov/Blast.cgi).
alignment
was
analyzed
SignalP
using
4.1
ClustalW
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111
version 6.0 [22].
112
2.3 Primary culture of ayu head kidney-derived MO/MΦ
113
Ayu head kidney-derived MO/MΦ were isolated as previously described [23], with
114
some modifications. Briefly, head kidneys were aseptically extracted, and dissociated
115
in RPMI 1640 (Invitrogen, Shanghai, China) supplemented with 2% fetal bovine
116
serum (FBS) (Invitrogen), penicillin (100 U/ml), streptomycin (100 mg/ml), and
117
heparin (20 U/ml). Cells were separated using Ficoll-Hypaque PREMIUM (1.077
118
g/ml) (GE Healthcare, New Jersey, USA) in combination with centrifugation
119
according to the manufacturer's instructions. Cells were then seeded onto 35-mm well
120
plates at a density of 2 × 106 cells per well and allowed to adhere overnight at 24°C in
121
an incubator with 5% CO2. The medium was changed to complete medium (4% ayu
122
serum, 6% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin), and cells were kept in
123
the incubator under the same conditions.
124
2.4 Sample preparation for PaCD302 mRNA expression analysis
125
Ayu tissues including liver, muscle, head kidney, brain, spleen, intestine, gill, MO/MΦ,
126
peripheral blood lymphocytes (PBLs) were collected from healthy fish for tissue
127
PaCD302 mRNA expression profile analysis. For pathologically correlated expression
128
analysis, fish were challenged by intraperitoneal injection with 1.2x104 colony
129
forming units (CFUs) live V. anguillarum (in 100 µl PBS) per fish, and PBS alone was
130
used as a control. Liver, head kidney, spleen, and PBLs samples were collected at 4, 8,
131
12, and 24 h post infection (hpi). Head kidney-derived MO/MΦ were purified as
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ACCEPTED MANUSCRIPT described in Section 2.3 and infected with live V. anguillarum at a multiplicity of
133
infection (MOI) of 10. Tissues were immediately snap-frozen in liquid nitrogen and
134
preserved in -80°C until subsequent use. PBLs and MO/MΦ were resuspended in
135
RNAiso (TaKaRa, Dalian, China) before RNA extraction.
136
2.5 Real-time quantitative PCR (qPCR) assay
137
Total RNA was extracted using RNAiso reagents (TaKaRa), followed by
138
deoxyribonuclease I digestion to eliminate genomic DNA. Specific primers for
139
PaCD302
140
AGCAAGAAGGTGTGGAAGGAT-3′
141
GAGGTCAGAAGAGAAGCCAC-3′, which was expected to amplify a 135 base pair
142
(bp)
143
5′-TCGTGCGTGACATCAAGGAG-3′
144
5′-CGCACTTCATGATGCTGTTG-3′, was used to amplify a 231-bp fragment from
145
β-actin, which is widely used as an internal control in ayu [8,19]. The qPCR reaction
146
was performed in an RT-Cycler™ Realtime Fluorescence Quantitative PCR
147
thermocycler (CapitalBio, Beijing, China) using SYBR premix Ex Taq (Perfect Real
148
Time) (TaKaRa). The reaction mixture was incubated for 5 min at 95°C, followed by
149
40 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. The mRNA expression of
150
PaCD302 was normalized to that of β-actin using the 2-∆∆Ct method [24].
151
2.6 Preparation of recombinant PaCD302 (rPaCD302)
152
The primers rPaCD302 (+): 5′-GGAATTCGATTGCCCTGCGGATGGG-3′ and
153
rPaCD302 (-): 5′-GCTCGAGCTGGCACACCACCCCATTC-3′ (underlined are the
Another
PaCD302-T
and
PaCD302-T
primer
pair,
(+):
5′-
(−):
5′-
pActin2
and
(+):
pActin2(−):
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ACCEPTED MANUSCRIPT EcoRI and XhoI sites, respectively) were used to amplify the sequence encoding the
155
extracellular region of PaCD302. Amplicons of the expected size were digested using
156
EcoRI and XhoI and cloned into the pET28a(+) vector. The recombinant plasmid
157
pET28a-PaCD302 was transformed into Escherichia coli BL21 (DE3). rPaCD302
158
was overexpressed as inclusion bodies, which were dissolved in an 8 M urea solution
159
and purified using a nickel-nitrilotriacetic acid (Ni-NTA) column (QIAGEN, Hilden,
160
Germany) at 4°C. Refolding of solubilized rPaCD302 using 8 to 2 M urea gradient
161
size-exclusion chromatography was performed on an XK 16/100 column packed with
162
Superdex 75 gel media (GE Healthcare) at 4°C. Peak fractions were pooled and
163
concentrated using a 10,000 NMWL spin filter (Millipore, Shanghai, China) at 4°C.
164
The eluted fractions were desalted on a Bio-Gel P-6 desalting column (Bio-Rad,
165
Shanghai, China), then lyophilized and stored at −80°C before use.
166
2.7 Antibody preparation and western blot assay
167
Antibody production was performed as previously reported [8]. Briefly, rPaCD302
168
emulsified with Freund’s incomplete adjuvant was used to immunize mice by
169
intraperitoneal injection once every seven days for a total of four injections. Control
170
mice were injected with complete Freund’s adjuvant. Whole blood was collected and
171
centrifuged to obtain sera. Anti-rPaCD302 IgG and mouse isotype IgG were purified
172
using protein G chromatography media (Bio-Rad).
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Western blot was performed to detect native PaCD302 in ayu MO/MΦ using the
174
prepared antibody. Briefly, total protein was extracted from MO/MΦ, resolved by
175
SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Mouse
ACCEPTED MANUSCRIPT 176
anti-PaCD302 antiserum was used as the primary antibody at a 1:1000 dilution,
177
followed by horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (1:5000) as
178
the
179
chemiluminescence (ECL) kit (Advansta, Menlo Park, USA). In order to determine
180
whether native PaCD302 is N-glycosylated, denatured proteins of ayu MO/MΦ were
181
treated with PNGase F (New England Biolabs, Beverly, USA) at 37°C overnight, and
182
analyzed by western blot.
183
2.8 Sugar binding assay
184
An indirect enzyme-linked immunosorbent assay (ELISA) was performed on high
185
affinity ELISA plates (Corning Costar, NY, USA) to detect the binding capacity of
186
rPaCD302 to five monosaccharides, L-fucose, D-galactose, D-glucose, D-mannose
187
and GlcNAc, and two polysaccharides, lipopolysaccharide (LPS) (E. coli 055: B5)
188
and peptidoglycan (PGN) (Staphylococcus aureus) (Sigma Chemical Co., St. Louis,
189
USA), according to previously described methods [8,25]. Briefly, each well of a
190
microplate was coated with 50 µl of sugar (80 µg/ml) in TBS (10 mM Tris-HCl,
191
pH7.5, 150 mM NaCl) and incubated overnight at 37°C. After heating at 60°C for 30
192
min, the plate was blocked with 50 µl of 1 mg/ml BSA per well for 2 h at 37°C, and
193
washed four times with TBS. rPaCD302 (0–100 µg/ml protein, diluted with TBS
194
containing 0.1 mg/ml of BSA) was added to each well and incubated for 3 h at 30°C.
195
After washing with TBS to remove free rPaCD302, 100 µl of anti-PaCD302 IgG
196
(1:2000 diluted) was added to each well and incubated for 1 h at 37°C. After washing
197
with TBS to remove free anti-PaCD302 IgG, 100 µl of peroxidase-conjugated goat
were
visualized
using
an
enhanced
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199
Stabilized 3, 3′, 5, 5′-tetramethylbenzidine (Sigma Chemical Co.) solution containing
200
hydrogen peroxide was added to each well, the plate was incubated at room
201
temperature and the absorbance of each well was measured using a SpectraMax® M3
202
multi-mode microplate reader (Molecular Devices, Sunnyvale, USA). The negative
203
control used TBS instead of rPaCD302. All experiments were repeated three times.
204
2.9 Bacterial binding and agglutination assay of rPaCD302
205
Three Gram-positive bacteria (Listeria monocytogenes, S. aureus, and Streptococcus
206
iniae) and seven Gram-negative bacteria (Aeromonas hydrophila, E. coli,
207
Edwardsiella tarda, Vibrio alginolyticus, V. anguillarum, Vibrio harveyi, and Vibrio
208
parahaemolyticus) were selected for the assay. Bacteria were cultured overnight to
209
analyze the binding capacity of rPaCD302 as previously described [25] in the
210
presence or absence of 10 mM CaCl2. After centrifugation at 6000 × g for 5 min, cell
211
pellets were collected, washed twice with TBS, then thoroughly resuspended in TBS.
212
Resuspended bacteria (500 µl, 2 × 108 cells/ml) were incubated with rPaCD302 (final
213
concentration 0.5 mg/ml) at room temperature for 30 min. Microorganisms were
214
pelleted, washed four times with TBS, and eluted with 7% SDS for 1 min. Cells
215
incubated with TBS only were used as a control. Pellets were subjected to SDS-PAGE,
216
and the presence of rPaCD302 was determined using western blot.
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Bacterial agglutination activity of rPaCD302 was assayed as previously described
218
[8]. Cells were harvested at the mid-logarithmic phase, and were resuspended to 2 ×
219
108 cells per ml in TBS. The microorganism/TBS solution (25 µl) was incubated with
ACCEPTED MANUSCRIPT 25 µl rPaCD302/TBS (200 µg/ml) at 28ºC for 1 h in the presence or absence of 10
221
mM CaCl2. Agglutination of bacteria was analyzed by a bright-field microscopy
222
OLYMPUS BH-2 (Olympus, Tokyo, Japan).
223
2.10 In vitro phagocytosis assay of ayu MO/MΦ
224
Logarithmic phase E. coli DH5α were labeled with fluorescein isothiocyanate (FITC)
225
(Sigma Chemical Co.) according to the manufacturer’s protocol, and FITC-labeled
226
bacteria were hereafter designated as E. coli-FITC. For neutralization analysis, ayu
227
MO/MΦ grown on cover slips in 35 mm plates (2 × 106 cells/well) were pre-incubated
228
with 200 µg/ml anti-PaCD302 IgG or isotype IgG for 30 min. PBS was used as a
229
control. E. coli-FITC was added at a MOI of 10, and cells were further incubated for
230
30 min. Trypan blue (0.4%) was used to quench the fluorescence that resulted from
231
particles that were outside of the cells or sticking to the cell surface. Engulfed bacteria
232
were examined using a Gallios flow cytometer (Beckman Coulter, Miami, USA) and
233
data were analyzed using FlowJo software. Relative mean fluorescence intensity (MFI)
234
of the IgG-treated group was expressed as fold-change relative to the value of the
235
no-bacteria control, and the value of the isotype IgG-treated group was assigned a unit
236
of 100.
237
2.11 Assay of MO/MΦ bactericidal activity
238
Ayu MO/MΦ were pre-incubated with 200 µg/ml anti-PaCD302 IgG or isotype IgG
239
for 30 min, and then infected with live V. anguillarum at a MOI of 10. MO/MΦ
240
phagocytosis of bacteria was allowed to proceed for 30 min at 24°C in an atmosphere
241
of 5% CO2, and noninternalized V. anguillarum were removed by washing with sterile
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ACCEPTED MANUSCRIPT PBS. One set of samples (the uptake group) was lysed in a 1% Triton X-100 solution
243
and plated onto solid thiosulfate citrate bile salts sucrose (TCBS) agar medium to
244
provide bacterial uptake values. The remaining set (the kill group) was incubated for a
245
further 1.5 h to allow bacterial killing to occur before cell lysis. After incubation at
246
30°C for 18 h, the number of colonies on each plate was counted. Bacterial survival
247
rate was determined by dividing the CFU of the kill group by that of the uptake group.
248
Three independent experiments were carried out.
249
2.12 Statistical analysis
250
All data were reported as mean ± SEM. Statistical analysis of the results was
251
conducted using one-way Analysis of Variance (ANOVA) with SPSS version 13.0
252
(SPSS Inc, Chicago, USA). P values less than 0.05 were considered statistically
253
significant.
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3. Results
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3.1 Cloning and sequence analysis of PaCD302
257
The cDNA sequence of PaCD302 was identified by a BLAST search and submitted to
258
the DDBJ/EMBL/GenBank databases under accession number JP773538. The cDNA
259
is 1893 nucleotides (nt) in length and contains a large open reading frame (ORF) of
260
726 nt, which is predicted to encode a 241 amino acid (aa) polypeptide with a
261
calculated molecular weight (MW) of 27.1 kDa and a pI of 4.69. PaCD302 is the
262
simplest type I transmembrane C-type lectin discovered to date; it contains a signal
263
peptide (aa 1-26), one CTLD (aa 29-163), and one short spacer followed by one
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ACCEPTED MANUSCRIPT transmembrane domain (aa 179-201) and one cytoplasmic tail (Fig. 1A). The
265
asparagine (N) residue at aa position 149 is predicted to be an N-glycosylated site (Fig.
266
1A). The PaCD302 CTLD contains 6 cysteine (C) residues, which is a typical feature
267
of a C-type lectin motif, forming an intramolecular disulfide bond-mediated C-type
268
lectin fold (Fig. 1A). Multiple alignment shows that PaCD302 CTLD is devoid of the
269
known amino acid residues essential for Ca2+-dependent sugar binding, i.e. the EPN
270
motif (for mannose binding), the QPD motif (for galactose binding), and the WND
271
motif (for Ca2+ binding) (Fig. 1B).
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Fig. 1. Multiple alignment of the amino acid sequences of PaCD302 and related
274
proteins from other vertebrates. (A) Comparison of amino acid sequences of
275
vertebrate CD302. Conserved cysteine residues are marked as “*”. The asparagine
276
residue, predicted to be an N-glycosylated residue, is marked as “
277
of CTLD sequences from human dendritic cell-specific intercellular adhesion
”. (B) Comparison
ACCEPTED MANUSCRIPT molecule-3-grabbing non-integrin (hDC-SIGN), human macrophage galactose
279
binding lectin (hMGL), ayu C-type lectin receptor C (PaCTLRC) and PaCD302. The
280
single and double underlines indicate the positions of the EP(N/Q)PD and WND
281
motifs, respectively. Threshold for shading was 70%; similar residues are marked with
282
a gray shadow, identical residues with a black shadow, and alignment gaps with “-”.
283
Accession numbers: ayu CD302, JP773538; black rockcod (Notothenia coriiceps)
284
CD302, XM_010782533; rainbow trout (Oncorhynchus mykiss) CD302, FR904281;
285
Japanese ricefish (Oryzias latipes) CD302, XM_004081729; large yellow croaker
286
(Larimichthys crocea) CD302, KQ042842; Nile tilapia (Oreochromis niloticus)
287
CD302, XM_005452988; zebrafish (Danio rerio) CD302, XM_002667634; human
288
(Homo sapiens) CD302, NM_014880; PaCTLRC, KP329196; hDC-SIGN, AF290886;
289
hMGL, BC039011.
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Sequence comparison showed that PaCD302 shared the highest aa identity (65%)
291
with CD302 from black rockcod. Phylogenetic tree analysis of amino acid sequences
292
showed that PaCD302 falls into the group of fish CD302, but is distant from other
293
smaller clusters (Fig. 2).
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Fig. 2. Phylogenetic analysis of amino acid sequences of CD302 using the
296
neighbor-joining method. Human CD302 was used as an outgroup to root the tree.
297
Values at the forks indicate the percentage of trees in which this grouping occurred
298
after bootstrapping (1,000 replicates; shown only when > 60%). Scale bar shows
299
number of substitutions per base. Accession numbers are listed in the legend of Fig. 1
300
and below: Mexican tetra (Astyanax mexicanus), XM_007230421; Atlantic salmon
301
(Salmo
302
XM_008328697; zebra mbuna (Maylandia zebra), XM_004557639; killfish
303
(Austrofundulus limnaeus), XM_014016681; Amazon molly (Poecilia formosa),
304
XM_007546599; spotted green pufferfish (Tetraodon nigroviridis), CAAE01015004;
305
tiger pufferfish (Takifugu rubripes), XM_003961965; northern pike (Esox lucius),
306
XM_010890847; southern platyfish (Xiphophorus maculatus), XM_005805321.
307
3.2 qPCR analysis of PaCD302 mRNA expression
308
qPCR was used to analyze the tissue mRNA expression profile of PaCD302 in healthy
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XM_014164142;
tongue
sole
(Cynoglossus
semilaevis),
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ACCEPTED MANUSCRIPT ayu. PaCD302 mRNA was constitutively expressed in all tissues tested, including
310
liver, heart, brain, spleen, gill, intestine, muscle, head kidney, PBLs, and MO/MΦ,
311
with the highest expression in the liver (Fig. 3A). Upon V. anguillarum infection,
312
PaCD302 mRNA expression was found to be upregulated (Fig. 3B-F). PaCD302
313
mRNA expression in the liver, spleen and PBLs was upregulated at 4 hpi, followed
314
thereafter by a decrease. The highest transcript levels of PaCD302 in the head kidney
315
occurred at 24 hpi (Fig. 3B-E). The most significant increase in PaCD302 transcript
316
levels was observed in the liver at 4 hpi (16.8-fold) (Fig. 3B-E).
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317 318
Fig. 3. qPCR analysis of PaCD302 transcripts in ayu tissues and cells. (A) mRNA
ACCEPTED MANUSCRIPT expression profiles of PaCD302 in healthy ayu tissues and cells. L: liver, H: heart, B:
320
brain, S: spleen, MO/MΦ: monocytes/macrophages, G: gill, I: intestine, M: muscle,
321
HK: head kidney, PBLs: peripheral blood lymphocytes. (B-F) Changes in PaCD302
322
mRNA expression in tissues and cells of the ayu immune system, following V.
323
anguillarum infection. Tissues and cells were collected at different time points
324
post-bacterial infection. PaCD302 mRNA expression was normalized to β-actin. Data
325
are expressed as mean ± SEM of results from four individual fish. * P < 0.05.
326
3.3 Prokaryotic expression, purification and antibody preparation of rPaCD302
327
The recombinant protein was successfully expressed in E. coli with a MW of 24.5
328
kDa in SDS-PAGE, which was 4.1 kDa larger than the MW calculated from sequence
329
data (20.4 kDa, comprising 15.7 kDa protein and 4.7 kDa His-tag) (Fig. 4A). The
330
aberrant SDS–PAGE behavior of PaCD302 may be due to mobility retardation of the
331
His-tag [26] and the reduced protein structure [12]. The overexpressed protein was
332
extracted from inclusion bodies, purified by affinity chromatography using an
333
Ni-NTA column, and refolded by 8 to 2 M urea gradient size-exclusion
334
chromatography (Fig. 4A). rPaCD302 was then used to immunize mice to produce the
335
antiserum. Using this antiserum, we determined that the MW of the native PaCD302
336
in MO/MΦ was about 32 kDa by western blot analysis (Fig. 4B), which was bigger
337
than that calculated from sequence data (27.1 kDa). After PNGase F digestion, the
338
MW of the native PaCD302 decreased to about 27 kDa, suggesting the existence of
339
post-translational N-glycosylation, as was previously reported for human CD302 [13].
340
Anti-rPaCD302 IgG was purified from antisera using protein G chromatography
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ACCEPTED MANUSCRIPT 341
media for subsequent use.
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Fig. 4. Prokaryotic expression of PaCD302 and western blot analysis. (A) 12%
345
SDS–PAGE analysis of bacterial lysates and purified rPaCD302. Lane M: protein
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marker; Lane 1: lysates of pET28a-PaCD302 transformed E. coli BL21 without IPTG
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induction; Lane 2: lysates of pET28a-PaCD302 transformed E. coli BL21 with IPTG
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induction; Lane 3: purified rPaCD302. (B) Western blot analysis of PaCD302 using
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antisera against rPaCD302. Lane 4: total protein extracted from ayu MO/MΦ; Lane 5:
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total protein extracted from ayu MO/MΦ, PNGase F digested; Lane 6: E. coli BL21
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lysates, negative control.
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3.4 Carbohydrate and bacteria binding capacity of rPaCD302
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We investigated the OD 450 values, which reflect the binding capacity of rPaCD302,
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using a carbohydrate binding assay. rPaCD302 was unable to bind five
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ACCEPTED MANUSCRIPT monosaccharides and two polysaccharides. Western blot analysis was used to
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determine the bacteria binding capacity, which revealed that rPaCD302 was able to
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bind all ten of the bacteria tested, in the presence of calcium ions. Binding of
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rPaCD302 to S. iniae and E. tarda was relatively weaker than it was to other bacteria
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(Fig. 5). However, rPaCD302 did not agglutinate all microorganisms tested (data not
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shown).
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Fig. 5. Western blot analysis of bacteria binding activity of rPaCD302 in the presence
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(A) or absence (B) of Ca2+ by using a specific antiserum to PaCD302. PC: rPaCD302
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used as a positive control, Lmo: L. monocytogenes, Sau: S. aureus, Sin: S. iniae, Ahy:
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A. hydrophila, Eco: E. coli, Eta: E. tarda, Val: V. alginolyticus, Van: V. anguillarum,
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Vha: V. harveyi, Vpa: V. parahaemolyticus.
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3.5 PaCD302 effect on phagocytosis and bacterial killing by ayu MO/MΦ
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Human PaCD302 has been reported to be involved in phagocytosis by human MΦ
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[13]. We therefore used the anti-PaCD302 IgG to block PaCD302 function in order to
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investigate whether it has an effect on phagocytosis and bacterial killing by ayu
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MO/MΦ. Compared to the isotype IgG group, the phagocytic activity of E. coli-FITC
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by anti-PaCD302 IgG-treated MO/MΦ decreased by 28.3% (Fig. 6A), while the
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ACCEPTED MANUSCRIPT survival rate of V. anguillarum increased from 54.49 ± 4.82% to 75.2 ± 5.49% (Fig.
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6B).
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Fig. 6. Effect of PaCD302 blockage on phagocytosis and bacterial killing by ayu
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MO/MΦ. Anti-PaCD302 IgG was used to block PaCD302 function on MO/MΦ,
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while isotype IgG was used as a control. (A) Flow cytometry analysis of E. coli-FITC
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phagocytosis by ayu MO/MΦ. Relative mean fluorescence intensity (MFI) of
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anti-PaCD302 IgG- or isotype IgG-treated group expressed as fold-change, and the
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value of isotype control was assigned a unit of 100. Data are mean ± SD of three
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repeats. *P < 0.05. (B) Killing of V. anguillarum by ayu MO/MΦ was measured using
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a CFU assay. Data are expressed as mean ± SD of three repeats. *P < 0.05.
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4. Discussion
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CTLRs comprise a heterogeneous group of transmembrane proteins that are able to
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recognize pathogens and in some cases engage signaling pathways that propagate into
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cells to elicit microbicidal or inflammatory responses [8,20,27]. In the present study,
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we identified for the first time a CD302 homologue in ayu. PaCD302 is predicted to
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be a type I transmembrane CTLR with a signal peptide, a CTLD, a short spacer
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followed by a transmembrane domain and a cytoplasmic domain. PaCD302 mRNA is
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widely expressed in ayu tissues and immune cells, and is especially abundant in the
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liver, which is similar to that reported for other fish CTLRs [7,9,28]. CD302 in
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mammalian leukocytes has been restricted to monocytes, macrophages, granulocytes,
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and dendritic cells. Liver contains blood and many immune cells such as
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tissue-resident mature macrophages-Kupffer cells. On the other hand, head
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kidney-derived MO/MΦ is in an immature state. Therefore, the mRNA expression
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levels of PaCD302 in several tissues, especially the liver, were higher than that in
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MO/MΦ. The transcript of PaCD302 in MO/MΦ was higher than that in PBLs is
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possibly caused by the percentage of cells expressing CD302. When ayu was infected
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by V. anguillarum, PaCD302 mRNA expression was upregulated in all tissues tested
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and in immune cells. These results suggest that PaCD302 should be involved in the
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innate immune response of ayu against bacterial infection.
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Unlike Ca2+-dependent CRDs found in prototypical CTLRs such as
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DC-SIGN/CD209, PaCD302 CTLD is devoid of the amino acid residues known to be
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have classic sugar binding capacity. In a previous report, human CD302 was shown to
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be unable to bind mannan, mannose, GlcNAc, and GalNAc [13]. In the present study,
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we confirmed that PaCD302 was not able to bind all tested saccharides, which is in
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agreement with that predicted from sequence analysis and that reported for human
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CD302. A number of CTLRs exist today for which only a few, and sometimes no,
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carbohydrate ligands have been identified [2]. The nonclassical CTLRs have evolved
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to bind sugar and nonsugar ligands without classic Ca2+ and sugar binding motifs, for
414
example, dectin-1 for β-glucan in a Ca2+-independent manner [29]. It is possible that
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PaCD302 has some alternative carbohydrate binding capacity like dectin-1. It is also
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possible that PaCD302 binds to protein ligands in the same manner that CD209a
417
binds to LECT2 [30] and that P2X7R binds to cathelicidin [18]. It will be interesting
418
to determine whether PaCD302 has a binding capacity for alternative carbohydrates or
419
other biological structures in the future.
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Agglutinating activity of lectins is a basic reaction against invading pathogens,
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and binding is the first step in this process [31]. In the present study, we tested the
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bacteria agglutination activity of rPaCD302 and showed that it was able to bind all ten
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bacteria tested, but no obvious agglutination was observed. In addition to bacterial
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saccharides like LPS and PGN, other PAMPs such as lipoproteins, flagellin, bacterial
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nucleic acid structures are also the possible ligands of CTLRs. For examples,
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DEC-205 could bind to plasminogen activator (PLA) on the surface of Yersinia pestis,
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which mediates bacterial attachment [32]. DC-SIGN could bind to Surface (S) layer A
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ACCEPTED MANUSCRIPT protein (SlpA) on the surface of Lactobacillus acidophilus [33]. In this study,
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PaCD302 did not bind to classical saccharides, but it did bind to many bacteria. If
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there are bacterial PAMPs recognized by PaCD302, they are likely to have a high pI
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and be rich in basic amino acids because the PaCD302 extracellular domain is rich in
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acidic amino acids. There have been reports of other C-type lectins that can only bind
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bacteria but show no bacterial agglutinating activity. For example, Pc-Lec1 and
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Pc-Lec2 from red swamp crayfish (Procambarus clarkia) did not agglutinate any of
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the Gram-positive and Gram-negative bacteria tested, but they were able to bind to
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them [34,35]. Thus, our result suggests that PaCD302 may serve as a “sensor,” which
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can recognize invading pathogens, bind to them, and then initiate an immune response,
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rather than agglutinating them.
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At present, it is known that fish possess a well-developed non-specific immune
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system and that phagocytosis plays an important role in their defense against
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microorganisms [36]. Due to the importance of this process, several studies have
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focused on CTLRs that mediate phagocytosis in fish. For example, mannose receptor
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(MR) from blunt bream carp (Megalobrama amblycephala) is involved in the
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macrophage engulfment of GFP-expressing E. coli, resulting in a respiratory burst,
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nitric oxide production and inflammatory cytokine secretion [37]. In our previous
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reports, another two CTLRs from ayu, PaCD209L and PaCTLRC, were shown to
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recognize pathogens and participate in phagocytosis and the bacterial killing process
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of ayu MO/MΦ [8,25]. In the present study, we observed that function-blocking of
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PaCD302 effectively inhibited phagocytosis and bacterial killing of ayu MO/MΦ,
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ACCEPTED MANUSCRIPT revealing that PaCD302 should also function as a PRR. A previous report on human
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CD302 showed that the effect of function-blocking on macrophage phagocytosis was
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about 8-fold less efficient than the effect of macrophage MR or DEC-205
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function-blocking on macrophage phagocytosis [13]. Our study also revealed that the
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effect of PaCD302 function-blocking on MO/MΦ phagocytosis of E. coli-FITC
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(approximately 71.7% of the control) was obviously less efficient than the effect of
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PaCD209L function-blocking (approximately 25.1% of the control) [25]. This is in
457
agreement with findings from mammals [13], and means that CD302 could also have
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a weaker influence than other CTLRs on macrophage phagocytosis in fish. Human
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CD302 was reported to colocalize with F-actin, suggesting it may play a role in cell
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adhesion and migration [13]. However, this remains to be confirmed in fish.
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In conclusion, we have identified the characteristics of a novel CD302 in ayu,
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revealing it to be an unconventional CTLR. PaCD302 mRNA was significantly
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upregulated upon bacterial challenge in all tissues tested. PaCD302 participated in the
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phagocytosis and bacterial killing processes of ayu MO/MΦ. This is the first report on
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the functional analysis of a fish CD302-like protein, and our data demonstrate that
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PaCD302 may play a role in the ayu immune response to bacterial infection.
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Acknowledgments
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This project was supported by the Program for the National Natural Science
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Foundation of China (31372555), the Research Project of Chinese Ministry of
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Education (213017A), Zhejiang Provincial Natural Science Foundation of China
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(LZ13C190001), and the KC Wong Magna Fund in Ningbo University.
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ACCEPTED MANUSCRIPT Highlights
> We characterized a novel CD302 (PaCD302) gene from ayu.
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> PaCD302 mRNA expression responded to Vibrio anguillarum infection. > PaCD302 did not bind seven common saccharides such as d-mannose, LPS and PGN.
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> PaCD302 bound to ten microorganisms, but did not agglutinate them.
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> PaCD302 regulated the phagocytic and bactericidal activities of MO/MΦ in vitro.