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Identification and characterisation of a novel small galectin in razor clam (Sinonovacula constricta) with multiple innate immune functions
Accepted Manuscript Identification and characterisation of a novel small galectin in razor clam (Sinonovacula constricta) with multiple innate immune functions Yuqi Bai, Donghong Niu, Yan Li, Yulin Bai, Tianyi Lan, Maoxiao Peng, Zhiguo Dong, Fanyue Sun, Jiale Li PII:
S0145-305X(18)30475-0
DOI:
https://doi.org/10.1016/j.dci.2018.10.015
Reference:
DCI 3284
To appear in:
Developmental and Comparative Immunology
Received Date: 14 September 2018 Revised Date:
27 October 2018
Accepted Date: 29 October 2018
Please cite this article as: Bai, Y., Niu, D., Li, Y., Bai, Y., Lan, T., Peng, M., Dong, Z., Sun, F., Li, J., Identification and characterisation of a novel small galectin in razor clam (Sinonovacula constricta) with multiple innate immune functions, Developmental and Comparative Immunology (2018), doi: https:// doi.org/10.1016/j.dci.2018.10.015. 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
Identification and characterisation of a novel small galectin in razor clam
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(Sinonovacula constricta) with multiple innate immune functions
3 Yuqi Bai a, Donghong Niu ab*, Yan Li a, Yulin Bai a, Tianyi Lan a, Maoxiao Peng a,
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Zhiguo Dong d, Fanyue Sun e, Jiale Li ac*
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a
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Ministry of Education, Shanghai Ocean University, Shanghai 201306, China
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b
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Shanghai Ocean University, Shanghai 201306, China
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Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,
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National Demonstration Center for Experimental Fisheries Science Education,
c
Shanghai Engineering Research Center of Aquaculture, Shanghai 201306, China
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d
Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute
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of Technology, Lianyungang 222005, China
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e
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Reconstructive Sciences, University of Connecticut Health Center. 263 Farmington
15
Avenue, Farmington, CT 06030
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*Corresponding author:
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Donghong Niu, Ph.D.,
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E-mail: [email protected]
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Tel.: +86 021 61900438
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Jiale Li, Ph.D.,
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E-mail: [email protected]
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Tel.: +86 021 61900566
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AC C
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Center for Regenerative Medicine and Skeletal Development, Department of
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ACCEPTED MANUSCRIPT Abstract: Galectins are lectins possessing an evolutionarily conserved carbohydrate
25
recognition domain (CRD) with affinity for β-galactoside. The key role played by
26
innate immunity in invertebrates has recently become apparent. Herein, a full-length
27
galectin (ScGal) was identified in razor clam (Sinonovacula constricta). The 528 bp
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open reading frame encodes a polypeptide of 176 amino acids with a single CRD and
29
no signal peptide. ScGal mRNA transcripts were mainly expressed in hemolymph and
30
gill, and were significantly up-regulated following bacterial challenge. Recombinant
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rScGal protein binds to and aggregates various bacteria, and has affinity for
32
peptidoglycan, lipoteichoic acid and
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hemocytes to phagocytose invading bacterial pathogens. ScGal is an important
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immune factor in innate immunity, and a small protein with multiple important
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functions.
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The protein also stimulates
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D-galactose.
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Keywords: Galectins; Innate immunity; Bacterial challenge; Agglutination;
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Phagocytosis; Sinonovacula constricta
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ACCEPTED MANUSCRIPT 1. Introduction
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Lectins mediate the recognition of pathogens via protein-carbohydrate interactions
42
(Robinson et al., 2006). Studies have shown that lectins are involved in a variety of
43
physiological functions including agglutination, proliferation, phagocytosis, signal
44
transduction, apoptosis, and autophagy (Eddie et al., 2009; Su et al., 2016; Sun et al.,
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2016). Animal lectins are classified according into five families according to the
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peptide sequence mediating sugar recognition; P-type, C-type, I-type, S-type
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(galectins), and pentraxins (S. H. Barondes et al., 1994).
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Galectins (Gals) are a phylogenetically conserved lectin family originally defined
49
in 1994, consisting of small soluble lectin proteins of ~130 amino acids with a
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carbohydrate recognition domain (CRD) that binds β-galactoside (Camby et al.,
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2006). To date, 15 galectins have been identified in mammals (Barondes et al., 1994;
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Cooper, 2002). Based on their domain organisation, mammalian galectins have been
53
classified into three types: ‘proto type’ members such as Gal1, 2, 5, 7, 11, 13 and 14
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have a single CRD; Gal 3 is the only ‘chimera type’ member and has a collagen
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repeating structure; ‘tandem-repeat type’ proteins contain two similar CRD domains
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(Barondes et al., 1994; Hirabayashi and Kasai, 1993). Gals lack a recognisable
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secretion signal sequence, and do not pass along the standard endoplasmic reticulum
59
(ER)/Golgi pathway, but are nevertheless secreted and can be found in the
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extracellular matrix (Liu et al., 2002).
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Gals play several roles in development, and in innate and adaptive immunity,
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including adhesion, regulation of cellular proliferation, and regulation of cell survival
63
(Ikemori et al., 2014; Liu and Rabinovich, 2010; Scott and Weinberg, 2002). Gals
64
reportedly act during development by binding endogenous glycans. However, in
65
recent studies, functions in innate and adaptive immunity have been discovered. Gals
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act as pattern recognition receptors (PRRs) that specifically bind to exogenous
67
glycans, thereby activating immune pathways (Vasta, 2012; Vasta et al., 2012).
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Pathogen-associated molecular patterns (PAMPs) are not present in higher animals,
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and are essential and unique to almost all microorganisms (Akira et al., 2006). In
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mammals, evidence indicates that Gal1 and its ligands act as a master regulator of
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immune responses including T-cell homeostasis and survival, T-cell immune
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disorders, and inflammation and allergies, as well as host-pathogen interactions
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(Camby et al., 2006).
The roles of lectins in the recognition of microbial glycans are particularly critical
75
in invertebrates, since these organisms lack immunoglobulins and rely solely in innate
76
immune mechanisms for recognition of potential microbial pathogens (Vasta et al.,
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1999). Furthermore, in invertebrates the molecular configuration of Gals is different
78
from those in mammals (Vasta et al., 2015). A galectin with four CRD tandem repeats
79
was first identified in eastern oyster (Crassostrea virginica), and found to act in
80
immune processes related to microbial recognition and phagocytosis (Tasumi and
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Vasta, 2007). Subsequently, four CRD galectins were identified in scallops and
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abalone, and shown to affect certain immune responses as PRRs (Maldonado-Aguayo
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et al., 2014; Song et al., 2011). In addition, galectins with two CRD tandem repeats
84
were identified in Hyriopsis cumingii, and found to agglutinate various bacteria and
85
stimulate phagocytosis in hemocytes (Bai et al., 2016). In Litopenaeus vannamei, a
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galectin with a single CRD has been identified (Hou et al., 2015).
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Razor clam (Sinonovacula constricta) is a mudflat shellfish of economic
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ACCEPTED MANUSCRIPT importance to aquaculture in China. Microbial pathogens can have a severely
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damaging effect on the clam aquaculture process. Herein, we identified and
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characterised a novel galectin (ScGal) in razor clam and investigated its role as a PRR
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in innate immunity. The findings may be of benefit to improving aquaculture
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production of this species in future.
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2. Materials and methods
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2.1. Experimental samples
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Adult razor clams (average body weight of 9.0 ± 0.4 g and average body length of 5.3
96
± 0.2 cm) were obtained from Yuejingyang Farm, Ninghai City, Zhejiang Province,
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China. Selected clams were kept in seawater at 25°C and 20‰ salinity for 1 week.
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Samples from seven healthy tissues (liver, gill, foot, hemolymph, mantle, gonad and
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siphon) were collected and immediately frozen in liquid nitrogen and stored at -80°C.
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2.2. Cloning the full-length ScGal cDNA
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Total RNA was extracted from the seven pooled tissue samples using an RNeasy Plus
102
kit (Qiagen, CA), and cDNA synthesis was performed using a PrimeScript RT reagent
103
kit (TaKaRa, Japan) according to the manufacturer's instructions. The cDNA sample
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was stored at -20°C.
A partial fragment of the ScGal gene was obtained from the cDNA library of S.
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constricta (Niu et al., 2013b). In order to determine the accuracy of the fragment, we
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used primer 5.0 software to design primers to amplify the target fragment. PCR
108
products were gel-purified using a MiniBest Agarose Gel DNA Extraction Kit Ver. 4.0
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(TaKaRa) then cloned into the pGEM-T Easy vector (TaKaRa). The resulting
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construct was transformed into competent Escherichia coli Top10 cells (Tiangen,
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China) and positive clones were sequenced by Sangon Biotech Company (Shanghai,
112
China). In order to obtain the full-length ScGal gene, we designed primers based on
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the verified fragments and used rapid-amplification of cDNA ends (RACE)
114
technology to amplify the ends of the fragments using a 5’-Full RACE kit (Clontech,
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USA) and a 3’-Full RACE kit (TaKaRa). All primers are listed in Table 1, and
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sequencing was performed as described above, followed by alignment splicing.
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2.3. Sequence analysis
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Sequence alignment was performed using the NCBI database and the BLAST tool
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(http://blast.ncbi.nlm.nih.gov/Blast.cgi). The open reading frame (ORF) of the ScGal
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gene was obtained using ORF finder (http://www.ncbi.nlm.nih.gov/gorf/orFigure.cgi).
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Protein
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(http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cg). Signal peptide analysis was
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performed
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three-dimensional structure of ScGal was predicted using SWISS-MODEL
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(http://swissmodel.expasy.org/). Amino acid sequences of galectins in other species
126
were obtained from GenBank. Multiple sequence alignment and phylogenetic
127
analyses were performed using the BioEdit Sequence Alignment Editor and MEGA
128
5.0 software, respectively.
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2.4. Real-time PCR analysis of ScGal mRNA expression
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A total of 500 ng of RNA for each sample was reverse-transcribed using a
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PrimeScript RT reagent Kit (TaKaRa) according to the manufacturer's instructions.
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Primer 5.0 software was used to design specific primers (Table 1) and optimisation
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was performed by melting curve analysis. Previous studies showed that 18S rRNA is
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a suitable housekeeping gene in S. constricta (Niu et al., 2013a, 2014). Quantitative
135
real-time PCR (qRT-PCR) was performed with 20 µl reactions containing 1.6 µl of
SignalP
predicted
by
conserved
domain
prediction
(http://www.cbs.dtu.dk/services/SignalP-2.0/).
The
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ACCEPTED MANUSCRIPT cDNA, 10 µl of 2× SYBR Premix Ex Taq, 0.8 µl of each primer, and 6.8 µl of ddH2O.
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Thermal cycling conditions included an initial denaturation at 95°C for 3 min,
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followed by 40 cycles at 95°C for 5 s, and 60°C for 30 s. The Fluorescent signal for
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the dissolution curve was measured, and relative expression levels were determined
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using the 2-∆∆Ct method.
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2.5. Responses of ScGal following bacterial challenge
142
Clams were kept in seawater at 25°C and 20‰ salinity for 1 week, and 300 adult
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clams (mean shell length of 5.3 ± 0.2 cm) were randomly divided into three groups of
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100 animals each. Members of each group were injected in the foot with 50 µl of
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Staphylococcus aureus or Vibrio anguillarum in phosphate-buffered saline (PBS;
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2×109 cells/ml), or PBS alone as a control. Hemolymph were collected at 0, 4, 8, 12,
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24, 48 and 72 h post-inoculation. RNA extraction, cDNA synthesis, qRT-PCR, and
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data analysis were performed as described in section 2.4.
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To reduce experimental error, extracting nine individuals at each time point to
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extract RNA from hemolymph, equally mixed every three RNA samples for reverse
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transcription of cDNA，and a total of three replicates for qRT-PCR. One-way
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ACCEPTED MANUSCRIPT analysis of variance was performed using SPSS software to determine significant
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differences between experimental and control groups. Differences were considered
154
significant at p <0.05 (*) and highly significant at p <0.01 (**).
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2.6. Expression and purification of recombinant rScGal
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Primers with restriction enzyme sites were designed based on both ends of the cDNA
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fragment encoding the mature ScGal protein (Table 1). Following amplification and
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gel purification, the fragment was digested with BamHI and XhoI (TaKaRa) at 37°C
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for 1 h, and the expression vector pET-28a (Novagen, Germany) was also digested.
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The digested fragment was then ligated with the vector overnight using T4 ligase
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(TaKaRa) and the resulting recombinant plasmid (pET-28a-ScGal) or empty pET-28a
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vector (negative control) were separately transformed into E. coli BL21 (DE3) cells.
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Nucleic acid sequencing was performed to confirm the successful introduction of the
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desired plasmid. Bacteria were cultured at 37°C until the absorbance at 600 nm
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reached 0.6−0.8. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was then added at a
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final concentration of 0.5 mM to induce protein expression, and culturing was
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continued at 18°C for 16 h. Bacteria were collected by centrifugation at 7,500 × g for
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ACCEPTED MANUSCRIPT 5 min, washed three times with PBS, resuspended in PBS containing 1% protease
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inhibitor, and sonicated on ice. The supernatant containing soluble protein was
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collected by centrifugation at 10,000 × g at 4°C for 10 min, and passed through a
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pre-equilibrated Ni2+-chelating Sepharose column (GE Healthcare, USA). The
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His-tagged target protein was purified and eluted with 200 mM imidazole in PBS (pH
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7.4).
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sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), passed through a Desalting
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Gravity Column (Sangon, China), eluted in PBS, and the protein concentration was
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determined using a bicinchroninic acid (BCA) kit (CWBIO, China).
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2.7. Binding of rScGal to microorganisms and carbohydrates
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Based on previous studies (Zhang et al., 2009), the binding activity of rScGal was
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tested against Gram-positive bacteria (S. aureus, Bacillus subtilis and Streptococcus
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agalactiae) and Gram-negative bacteria (Aeromonas hydrophila, E. coli and V.
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anguillarum). Carbohydrates were added to inhibit this process to investigate the
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binding of rScGal to bacterial surface glycans. Briefly, an overnight culture of
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bacteria was centrifuged at 7,500 × g for 10 min, washed twice with PBS, then
eluted
protein
was
analysed
by
12%
sodium
dodecyl
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ACCEPTED MANUSCRIPT thoroughly resuspended in PBS to an OD600 of 1.0. Purified recombinant protein (5
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µg) was then incubated with 500 µl of bacterial suspension with slight shaking for 20
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min at room temperature. Bacteria were centrifuged, washed four times with PBS, and
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eluted with 7% SDS for 1 min. Eluted proteins were resuspended in SDS-PAGE
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loading buffer and separated by 12% SDS-PAGE, followed by western blotting
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analysis using anti-His antibody. Subsequently,
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lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) were
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separately incubated with recombinant protein (5 µg) for 1 h at room temperature (50
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µl of 1 mg/ml for each carbohydrate). In the control group, recombinant protein was
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incubated with PBS alone. Finally, 500 µl of V. anguillarum suspension was added
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and the binding assay was performed as described above.
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2.8. Bacterial agglutination assay
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Gram-positive bacteria (S. aureus, B. subtilis and S. agalactiae) and Gram-negative
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bacteria (A. hydrophila, E. coli and V. anguillarum) were separately cultured
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overnight, collected by centrifugation, washed three times with PBS, and resuspended
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in 0.1 M Na2CO3 (pH 9.5) to a final concentration of 109 cells/ml. Fluorescein
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maltose,
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ACCEPTED MANUSCRIPT isothiocyanate (FITC; Solarbio, China) was added at a final concentration at 0.5
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mg/ml and incubated for 30 min at room temperature to ensure thorough labelling.
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After three washes to completely remove unlabelled FITC, bacteria were resuspended
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in PBS to a density of 109 cells/ml. Next, 20 µl of rScGal (100 µg/ml) was mixed with
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labelled bacteria and incubated for 1 h at room temperature with gentle shaking to
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ensure mixing, after which agglutination was observed using a fluorescence
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microscope (Leica, Germany).
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2.9. Effect of rScGal on phagocytosis in hemocytes
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Labelling of V. anguillarum and S. aureus with FITC was performed as described in
209
Section 2.8. Fresh hemocytes from clams were washed three times with PBS by
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centrifugation at 1000 × g for 5 min and diluted to 1×107 cells/ml using a flow
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cytometer. A 200 µl sample of bacterial suspension (108 cells/ml) was mixed with 180
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µl of hemocytes suspension and 20 µl of rScGal in a 1.5ml centrifuge tube. In the
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negative control, rScGal was replaced with bovine serine albumin (BSA) and PBS.
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Samples were then incubated in the dark at room temperature, mixed once every 5
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min, and analysed using a flow cytometer.
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ACCEPTED MANUSCRIPT 3. Results
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3.1. Characterisation of the ScGal sequence
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A galectin gene fragment was identified from the cDNA library of S. constricta (Niu
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et al., 2013b), and the full-length cDNA sequence was obtained and designated ScGal.
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It comprises 1066 bp with a 531 bp coding sequence (CDS) including a stop codon,
221
an 84 bp 5′ untranslated region (UTR), and a 451 bp 3′ UTR with a predicted
222
polyadenylation signal sequence (1026AATAAA1031) and a poly(A) tail. The ORF
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encodes a protein of 176 amino acid residues without a signal peptide, with a
224
calculated molecular mass of 19.77 kDa and the theoretical isoelectric point of 5.65.
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The sequence has only one CRD domain (residues 48−174), and this is the Gal with a
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single CRD among known aquatic mollusc sequences (Fig. S1).
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Multiple sequence alignment of ScGal with orthologs from aquatic molluscs
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revealed relatively high sequence conservation (Fig. S2), and some conservation with
229
vertebrates (Orycteropus afer afer) and arthropods (Pteropus alecto).
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To evaluate the molecular phylogeny of ScGal, a phylogenetic tree was
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constructed based on the sequences of galectin orthologs from different taxa using the
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ACCEPTED MANUSCRIPT neighbour-joining method (Fig. S3). The tree forms two distinct clusters for
233
invertebrates and vertebrates, and among invertebrates, ScGal is most closely related
234
to galectin-2 from Mytilus galloprovincialis.
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Prediction of the protein structure by SWISS-MODEL based on human galectin-4
236
(40.15% identity) showed that ScGal consists of a β-sandwich structure with two
237
anti-parallel β-sheet bundles. β‐strands F1−F5 form the convex side of the β‐sandwich
238
that is related to secretion (Fig. S4A). The concave side of the β‐sandwich comprises
239
β‐strands S1−S6 and contains the carbohydrate binding site (Fig. S4B), which is well
240
conserved amongst galectin family members (Bum-Erdene et al., 2015).
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3.2. Real-time PCR analysis of ScGal mRNA expression in different tissues
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Constitutive expression of ScGal was examined in different tissues of adult clams
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(Fig. 1A). The mRNA transcript of ScGal is highly expressed in hemolymph and gill
244
(p <0.05), with the highest in hemolymph. Whereas expression in mantle, foot,
245
siphon, liver and gonad was comparatively low.
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3.3. Expression of ScGal following bacterial challenge
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As shown in Fig. 1B, at several hours after injection of S. aureus or V. anguillarum,
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ACCEPTED MANUSCRIPT ScGal expression was significantly enhanced compared with the control group. In the
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S. aureus group, expression peaked at 12 h after injection (>3-fold), whereas
250
expression in the V. anguillarum group responded more slowly after injection and
251
reached a 3-fold increase at 72 h.
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3.4. Expression and purification of rScGal
253
To characterise the biological functions of ScGal, E. coli BL21 (DE3) cells
254
harbouring the pET-28a-ScGal construct were cultured, and rScGal was mainly
255
expressed as in soluble form and accumulated in the supernatant (Fig. 2). SDS-PAGE
256
yielded a distinct band with a molecular weight of ~26 kDa, in accordance with the
257
predicted molecular mass of the fusion protein. The recombinant rScGal fusion was
258
purified by affinity chromatography, and a final concentration of ~1.0 mg/ml was
259
measured using a BCA kit.
260
3.5. Binding activity of rScGal
261
The six bacterial strains were separately incubated with rScGal, washed thoroughly to
262
remove unbound protein (Fig. 3A), eluted with 7% SDS, and the eluate was analysed
263
by western blotting. As shown in Fig. 3B, rScGal bound to all six bacterial strains,
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ACCEPTED MANUSCRIPT with no obvious differences between species. Next, we pre-incubated rScGal
265
separately with six different types of carbohydrates, then with V. anguillarum cells. As
266
shown in Fig. 3C, compared with the control group, all treatments inhibited binding
267
of recombinant protein to bacteria to some extent. PGN showed the strongest
268
inhibition, followed by
269
maltose had a slight effect.
270
3.6. Effect of rScGal on agglutination of bacteria
271
FITC-labelled bacteria exhibited green fluorescence under a fluorescence microscope
272
(Fig. 4). However, after incubation with rScGal, all six bacterial strains exhibited
273
agglutination, with V. anguillarum showing the strongest agglutination.
274
3.7. Effect of rScGal on phagocytosis in hemocytes
275
Flow cytometry was used to measure samples (5 µl), and the results were drawn on a
276
scatter plot. To exclude the influence of fluorescence intensity in the area where
277
bacteria are located, the area where remaining hemocytes are located was defined as
278
Gate A. Subsequently, fluorescence data for each experimental group were limited to
279
Gate A (Fig. 5A). In addition, in the fluorescence intensity graph, based on untreated
and LTA. By contrast, LPS,
D-mannose
and
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ACCEPTED MANUSCRIPT hemocytes, we defined phagocytic parts of cells (Fig. 5B), and the fluorescence
281
intensity was positively correlated with the number of phagocytic bacteria. Phagocytic
282
ability (PA) is expressed as the proportion of hemocytes engaged in phagocytosis in
283
all cells. All experiments were repeated in triplicate, and the results are shown in the
284
marker section of the scatter plot (Fig. 5C).
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Statistical analysis of the data showed that the PA of hemocytes was stronger
286
following rScGal treatment than in the BSA-treated and untreated groups for both S.
287
aureus and V. anguillarum (Fig. 6). One-way analysis of variance was performed
288
using SPSS software to determine significant differences (p <0.05) between
289
experimental and control groups.
290
4. Discussion
291
Galectins are a large family of evolutionarily conserved proteins that contain one or
292
more CRDs with binding specificity for β-galactoside residues. In recent years, many
293
studies have shown that galectin activity is non-Ca2+-dependent, and plays a key role
294
in biological innate immune activities, especially in invertebrates that lack adaptive
295
immunity (Schulenburg et al., 2007; Wang and Zhao, 2004). In mammals, galectins
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ACCEPTED MANUSCRIPT are divided into three types; proto, chimera, and tandem repeat’. However, in aquatic
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molluscs, galectins are classified as 1-CRD, 2-CRD, 4-CRD, and chimeric ‘GREP’
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proteins (Vasta et al., 2015). In the present study, the full-length cDNA of a galectin
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was
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carboxyl-terminal CRD, and was hence classified as 1-CRD member. Amino acid
301
residues in the dimerisation interface are highly conserved, suggesting that this
302
galectin may play a role in the crosslinking of glycoprotein receptors and subsequent
303
cellular signalling events (Vladoiu et al., 2015). ScGal lacks a typical secretory signal
304
peptide,
305
ER/Golgi-independent pathway (Cha et al., 2015; Shi et al., 2014).
from
razor
clam
(Sinonovacula
constricta)
with
only
one
it
might
be
secreted
to
the
extracellular
space
via
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ScGal mRNA transcripts were detected in all tested tissues (Fig. 1A), and
307
expression was highest in hemolymph, followed by gill. Invertebrate hemolymph are
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involved in various aspects of surveillance and cellular immune responses (Johansson
309
et al., 2000), while gills are the first line of defence against aquatic microbial invasion
310
(Ellis, 2001). Therefore, hemolymph were selected for studying mRNA expression of
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ScGal following microbial invasion. The results revealed rapid up-regulation that
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ACCEPTED MANUSCRIPT peaked at 12 h after S. aureus challenge, whereas ScGal mRNA transcripts increased
313
gradually after challenge with V. anguillarum, and reached 3-fold up-regulation at 72
314
h. This indicates that ScGal is involved in acute immune responses against certain
315
bacteria (such as S. aureus). The fact that expression was significantly up-regulated
316
with both bacteria indicates broad resistance to pathogens, implying that ScGal is an
317
important immune factor. Similarly, Gal in Argopecten irradians is widely expressed
318
in various tissues, and is highly expressed in hemolymph after challenge with V.
319
anguillarum and Pichia pastoris (Song et al., 2011), and in Eriocheir sinensis, EsGal
320
acts as an acute protein-mediated immune response (Wang et al., 2016). This
321
immunological activity in different species further demonstrates the pivotal role of
322
galectins in innate immunity in invertebrates.
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As PRRs, lectins activate immune responses by recognising pathogens through
324
their unique CRDs (Liu, 2005). In Amphioxus, the AmphiITLN-like protein exhibits
325
preferential binding to PGN of Gram-positive bacteria (Yan et al., 2012). In A.
326
irradians, AiGal binds to a variety of bacteria, resulting in varying degrees of
327
agglutination (Song et al., 2010). Similarly, in the present study, ScGal showed strong
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We pre-incubated rScGal with various carbohydrates prior to binding to bacteria to
330
explore their inhibitory effects. The results indicate that rScGal binds PGN most
331
strongly, followed by D-galactose and LTA, and the other tested carbohydrates bound
332
weakly. PGN is the main component of many bacterial cell walls, and PGN in the cell
333
wall of Gram-positive bacteria (G+) accounts for ~50% of the dry weight, while PGN
334
in the cell wall of Gram-negative bacteria (G-) accounts for only ~10%. LTA is a
335
specific component of Gram-positive bacteria (Dziarski, 2003; Schleifer and Kandler,
336
1973; Wicken and Knox, 1975). The ability of ScGal to bind and aggregate three
337
Gram-positive and three Gram-negative bacterial species in this study was mainly
338
attributed too binding PGN, LTA and D-galactose on the bacterial surface.
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It has been reported that lectins are key factors in phagocytosis (Sharon, 1984), a
340
process associated with the ability of lectin receptors to recognise antigens (Vogel et
341
al., 1980). To test whether ScGal affects phagocytosis in hemocytes, we tested two
342
bacterial strains to investigate the phagocytic rate of hemocytes with FITC-labelled
343
bacteria after incubation with rScGal. The phagocytic ability of blood cells was
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ACCEPTED MANUSCRIPT significantly enhanced against both strains after rScGal was added. Thus, ScGal not
345
only acts as a PRR in the process of immune recognition, but also participates in the
346
pathogen clearance process.
347
5. Conclusion
348
A full-length galectin (ScGal) was identified in S. constricta. ScGal was highly
349
expressed in hemolymph, and strongly up-regulated after bacterial challenge.
350
Recombinant rScGal protein activated the immune response, agglutinated bacteria,
351
and stimulated the phagocytic action of hemocytes to enhance antigen clearance.
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Acknowledgements
354
This work was supported by the National Natural Science Foundation of China (grant
355
number 31472278), the National High Technology Research and Development
356
Program of China (863 Program; grant number 2012AA10A400-3), and the Shanghai
357
Universities Knowledge Service Platform (grant number ZF1206).
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Competing interests
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ACCEPTED MANUSCRIPT 360
The authors have no competing interests to declare.
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Tables
Table 1 Primers used to study the ScGal gene Sequence (5′-3′)
Comment
ScGal-F1
TTATTACCCGACAGACACTTAGC
Amplification of an
ScGal-R1
TGTGGAGGTTACAGTGTGGTG
ScGal-R2
TTCCGGTCGTGGGTCTGGAGTTTG
UPM
CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACG
Primer
ScGal gene fragment 5′RACE (ScGal)
SC
CAGAGT
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cDNA cloning
CGAAGGGCACATGCGGATTGAAGG
UPM
CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACG
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ScGal-R3
CAGAGT
CACAAGGGACAAGAGAAACGCACA
OUTER
TACCGTCGTTCCACTAGTGATTT
ScGal-F3
GTTATGAAGTCGGGTGTTTGCTATGG
INNER
CGCGGATCCTCCACTAGTGATTTCACTATAGG
qPCR analysis
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ScGal-F2
CACGACCGGAAATCATTGCT
ScGal-R4
CATGCGGATTGAAGGTGGTT
18S-F
TCGGTTCTATTGCGTTGGTTTT
18S-R
CAGTTGGCATCGTTTATGGTCA
expressiona ScGal-F5 ScGal-R5 a
AC C
Protein
3′RACE (ScGal)
3′RACE (ScGal)
RT-PCR (ScGal)
RT-PCR (Control)
EP
ScGal-F4
5′RACE (ScGal)
CGCGGATCCGCGATGTCAAGTAACCAAACTCCAGA
Construction
CCGCTCGAGCGGGTACGCCACCTTCGTCAGT
vectors
BamHI and XhoI sites are underlined. 492 493
32
of
ACCEPTED MANUSCRIPT Figure legends
495
Fig. 1. (A) Expression of ScGal mRNA in seven adult tissues determined by
496
qRT-PCR. Expression levels are relative to those in gonad tissue. Results are
497
representative of three independent experiments. Bars represent the mean ± standard
498
error (SE; n = 3) for each tissue. An asterisk above bars denotes a significant
499
difference (p <0.01) between tissues. (B) Expression profiles of ScGal mRNA in
500
hemocytes following challenge with Vibrio anguillarum or Staphylococcus aureus.
501
Clams injected with phosphate-buffered saline (PBS) were used as controls.
502
Differences between PBS and pathogen infection groups are shown (*p <0.05) and
503
(**p <0.01).
SC
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504
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Fig. 2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
506
analysis of the rScGal protein. Lane M, markers (25 kDa); lane 1, supernatant of
507
Escherichia coli cells harbouring the pET28a-ScGal construct after ultrasonication.
508
lane 2, purified rScGal protein.
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509
33
ACCEPTED MANUSCRIPT Fig. 3. (A) Binding of recombinant ScGal to S. aureus. Purified recombinant ScGal
511
was incubated with S. aureus cells in PBS at room temperature for 20 min. After
512
centrifugation to separate unbound protein and washing four times with PBS, bound
513
protein was eluted with 7% SDS for 1 min. Recombinant ScGal was detected using
514
antiserum against ScGal. Lane 1, wash solution from S. aureus cells (PBS controls);
515
lane 2, unbound protein; lanes 3−6, wash solutions from S. aureus cells incubated
516
with ScGal; lane 7, protein eluted with 7% SDS. (B) Binding of recombinant ScGal to
517
microorganisms. Recombinant ScGal was incubated with microorganisms as
518
described in panel A, and protein eluted with 7% SDS was detected by
519
immunoblotting with antiserum against ScGal. (C) Inhibition of the bacterial binding
520
activity of rScGal by carbohydrates. Different carbohydrates were added to test
521
whether they could inhibit binding to bacterial cells.
SC
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522
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510
523
Fig. 4. Agglutinating activity of rScGal against FITC-labelled V. anguillarum, E. coli,
524
Aeromonas hydrophila, S. aureus, Streptococcus agalactiae and Bacillus subtilis. PBS
525
was incubated with bacteria as a negative control.
34
ACCEPTED MANUSCRIPT 526
Fig. 5. Flow cytometry analysis of phagocytosis by hemocytes following exposure to
528
ScGal-treated, BSA-treated, or PBS-treated V. anguillarum or S. aureus. (A) Scatter
529
plots for V. anguillarum alone (left panel) and V. anguillarum + hemocytes. The area
530
where hemocytes are located is defined as Gate A. (B) Graph showing the
531
fluorescence intensity of hemocytes alone, revealing parts involved in phagocytosis.
532
(C) Phagocytosis in hemocytes exposed to PBS-, BSA- or ScGal-treated bacteria.
533
Results are averages of three independent experiments.
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Fig. 6. Analysis of phagocytosis after treatment with rScGal (or a negative control).
536
Differences between groups are shown (*p <0.05; **p <0.01).
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ACCEPTED MANUSCRIPT Figures
539
Fig.1.
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540
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541
36
ACCEPTED MANUSCRIPT Fig.2.
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542
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ACCEPTED MANUSCRIPT Fig.3.
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545
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546
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ACCEPTED MANUSCRIPT Fig.4.
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547
548 549
39
ACCEPTED MANUSCRIPT Fig.5.
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550
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ACCEPTED MANUSCRIPT Fig.6.
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ACCEPTED MANUSCRIPT 556 557
Supplementary data
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Fig. S1. Detection of putative conserved domains of Sinonovacula constricta ScGal
560
using the NCBI conserved domain database (CDD). The C-terminus (residues
561
48−174) is a carbohydrate recognition domain (CRD). Residues in the dimerisation
562
interface and sugar binding pocket are indicated by triangles.
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559
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42
564
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Fig. S2. Multiple sequence alignment ScGal from Sinonovacula constricta and related
566
proteins from various species using sequences obtained from the NCBI database.
567
Conserved amino acid residues are shaded dark grey, and similar amino acids are
568
shaded light grey. Residues involved in the dimerisation interface are in red boxes,
569
and the CRD is in a black box.
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Fig. S3. Phylogenetic tree showing homology between ScGal and galectins from
572
various vertebrates and invertebrates. The tree was constructed using a
573
ClustalW-generated multiple sequence alignment of amino acid sequences using the
574
neighbour-joining method in MEGA 5. The topological stability of trees was
575
evaluated by 10,000 bootstrapping replications. Numbers at branches indicate
576
bootstrap values (%).
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Fig. S4. The predicted 3D structure of ScGal. The protein fold consists of a
579
β-sandwich with two anti-parallel β-sheet bundles. (A) β‐strands F1−F5 form the
580
convex side of the β‐sandwich. (B) The concave side of the β‐sandwich comprises
581
β‐strands S1−S6. A bound lactose molecule in shown stick representation (grey
582
carbons and blue oxygens).
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ACCEPTED MANUSCRIPT Highlights
- A single CRD galectin was identified in razor clam (Sinonovacula constricta)
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- ScGal mRNA is mainly expressed in hemolymph and gill
- ScGal expression is up-regulated following bacterial challenge
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- ScGal aggregates bacteria, and binds PGN, LTA and D-galactose
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- ScGal also stimulates hemocytes to phagocytose invading bacterial pathogens
S0145-305X(18)30475-0
DOI:
https://doi.org/10.1016/j.dci.2018.10.015
Reference:
DCI 3284
To appear in:
Developmental and Comparative Immunology
Received Date: 14 September 2018 Revised Date:
27 October 2018
Accepted Date: 29 October 2018
Please cite this article as: Bai, Y., Niu, D., Li, Y., Bai, Y., Lan, T., Peng, M., Dong, Z., Sun, F., Li, J., Identification and characterisation of a novel small galectin in razor clam (Sinonovacula constricta) with multiple innate immune functions, Developmental and Comparative Immunology (2018), doi: https:// doi.org/10.1016/j.dci.2018.10.015. 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
Identification and characterisation of a novel small galectin in razor clam
2
(Sinonovacula constricta) with multiple innate immune functions
3 Yuqi Bai a, Donghong Niu ab*, Yan Li a, Yulin Bai a, Tianyi Lan a, Maoxiao Peng a,
5
Zhiguo Dong d, Fanyue Sun e, Jiale Li ac*
6
a
7
Ministry of Education, Shanghai Ocean University, Shanghai 201306, China
8
b
9
Shanghai Ocean University, Shanghai 201306, China
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4
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Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,
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National Demonstration Center for Experimental Fisheries Science Education,
c
Shanghai Engineering Research Center of Aquaculture, Shanghai 201306, China
11
d
Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute
12
of Technology, Lianyungang 222005, China
13
e
14
Reconstructive Sciences, University of Connecticut Health Center. 263 Farmington
15
Avenue, Farmington, CT 06030
16
*Corresponding author:
17
Donghong Niu, Ph.D.,
18
E-mail: [email protected]
19
Tel.: +86 021 61900438
20
Jiale Li, Ph.D.,
21
E-mail: [email protected]
22
Tel.: +86 021 61900566
23
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10
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Center for Regenerative Medicine and Skeletal Development, Department of
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ACCEPTED MANUSCRIPT Abstract: Galectins are lectins possessing an evolutionarily conserved carbohydrate
25
recognition domain (CRD) with affinity for β-galactoside. The key role played by
26
innate immunity in invertebrates has recently become apparent. Herein, a full-length
27
galectin (ScGal) was identified in razor clam (Sinonovacula constricta). The 528 bp
28
open reading frame encodes a polypeptide of 176 amino acids with a single CRD and
29
no signal peptide. ScGal mRNA transcripts were mainly expressed in hemolymph and
30
gill, and were significantly up-regulated following bacterial challenge. Recombinant
31
rScGal protein binds to and aggregates various bacteria, and has affinity for
32
peptidoglycan, lipoteichoic acid and
33
hemocytes to phagocytose invading bacterial pathogens. ScGal is an important
34
immune factor in innate immunity, and a small protein with multiple important
35
functions.
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The protein also stimulates
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D-galactose.
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Keywords: Galectins; Innate immunity; Bacterial challenge; Agglutination;
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Phagocytosis; Sinonovacula constricta
39
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41
Lectins mediate the recognition of pathogens via protein-carbohydrate interactions
42
(Robinson et al., 2006). Studies have shown that lectins are involved in a variety of
43
physiological functions including agglutination, proliferation, phagocytosis, signal
44
transduction, apoptosis, and autophagy (Eddie et al., 2009; Su et al., 2016; Sun et al.,
45
2016). Animal lectins are classified according into five families according to the
46
peptide sequence mediating sugar recognition; P-type, C-type, I-type, S-type
47
(galectins), and pentraxins (S. H. Barondes et al., 1994).
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Galectins (Gals) are a phylogenetically conserved lectin family originally defined
49
in 1994, consisting of small soluble lectin proteins of ~130 amino acids with a
50
carbohydrate recognition domain (CRD) that binds β-galactoside (Camby et al.,
51
2006). To date, 15 galectins have been identified in mammals (Barondes et al., 1994;
52
Cooper, 2002). Based on their domain organisation, mammalian galectins have been
53
classified into three types: ‘proto type’ members such as Gal1, 2, 5, 7, 11, 13 and 14
54
have a single CRD; Gal 3 is the only ‘chimera type’ member and has a collagen
55
repeating structure; ‘tandem-repeat type’ proteins contain two similar CRD domains
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57
(Barondes et al., 1994; Hirabayashi and Kasai, 1993). Gals lack a recognisable
58
secretion signal sequence, and do not pass along the standard endoplasmic reticulum
59
(ER)/Golgi pathway, but are nevertheless secreted and can be found in the
60
extracellular matrix (Liu et al., 2002).
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Gals play several roles in development, and in innate and adaptive immunity,
62
including adhesion, regulation of cellular proliferation, and regulation of cell survival
63
(Ikemori et al., 2014; Liu and Rabinovich, 2010; Scott and Weinberg, 2002). Gals
64
reportedly act during development by binding endogenous glycans. However, in
65
recent studies, functions in innate and adaptive immunity have been discovered. Gals
66
act as pattern recognition receptors (PRRs) that specifically bind to exogenous
67
glycans, thereby activating immune pathways (Vasta, 2012; Vasta et al., 2012).
68
Pathogen-associated molecular patterns (PAMPs) are not present in higher animals,
69
and are essential and unique to almost all microorganisms (Akira et al., 2006). In
70
mammals, evidence indicates that Gal1 and its ligands act as a master regulator of
71
immune responses including T-cell homeostasis and survival, T-cell immune
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disorders, and inflammation and allergies, as well as host-pathogen interactions
73
(Camby et al., 2006).
The roles of lectins in the recognition of microbial glycans are particularly critical
75
in invertebrates, since these organisms lack immunoglobulins and rely solely in innate
76
immune mechanisms for recognition of potential microbial pathogens (Vasta et al.,
77
1999). Furthermore, in invertebrates the molecular configuration of Gals is different
78
from those in mammals (Vasta et al., 2015). A galectin with four CRD tandem repeats
79
was first identified in eastern oyster (Crassostrea virginica), and found to act in
80
immune processes related to microbial recognition and phagocytosis (Tasumi and
81
Vasta, 2007). Subsequently, four CRD galectins were identified in scallops and
82
abalone, and shown to affect certain immune responses as PRRs (Maldonado-Aguayo
83
et al., 2014; Song et al., 2011). In addition, galectins with two CRD tandem repeats
84
were identified in Hyriopsis cumingii, and found to agglutinate various bacteria and
85
stimulate phagocytosis in hemocytes (Bai et al., 2016). In Litopenaeus vannamei, a
86
galectin with a single CRD has been identified (Hou et al., 2015).
87
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Razor clam (Sinonovacula constricta) is a mudflat shellfish of economic
5
ACCEPTED MANUSCRIPT importance to aquaculture in China. Microbial pathogens can have a severely
89
damaging effect on the clam aquaculture process. Herein, we identified and
90
characterised a novel galectin (ScGal) in razor clam and investigated its role as a PRR
91
in innate immunity. The findings may be of benefit to improving aquaculture
92
production of this species in future.
93
2. Materials and methods
94
2.1. Experimental samples
95
Adult razor clams (average body weight of 9.0 ± 0.4 g and average body length of 5.3
96
± 0.2 cm) were obtained from Yuejingyang Farm, Ninghai City, Zhejiang Province,
97
China. Selected clams were kept in seawater at 25°C and 20‰ salinity for 1 week.
98
Samples from seven healthy tissues (liver, gill, foot, hemolymph, mantle, gonad and
99
siphon) were collected and immediately frozen in liquid nitrogen and stored at -80°C.
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2.2. Cloning the full-length ScGal cDNA
101
Total RNA was extracted from the seven pooled tissue samples using an RNeasy Plus
102
kit (Qiagen, CA), and cDNA synthesis was performed using a PrimeScript RT reagent
103
kit (TaKaRa, Japan) according to the manufacturer's instructions. The cDNA sample
6
ACCEPTED MANUSCRIPT 104
was stored at -20°C.
A partial fragment of the ScGal gene was obtained from the cDNA library of S.
106
constricta (Niu et al., 2013b). In order to determine the accuracy of the fragment, we
107
used primer 5.0 software to design primers to amplify the target fragment. PCR
108
products were gel-purified using a MiniBest Agarose Gel DNA Extraction Kit Ver. 4.0
109
(TaKaRa) then cloned into the pGEM-T Easy vector (TaKaRa). The resulting
110
construct was transformed into competent Escherichia coli Top10 cells (Tiangen,
111
China) and positive clones were sequenced by Sangon Biotech Company (Shanghai,
112
China). In order to obtain the full-length ScGal gene, we designed primers based on
113
the verified fragments and used rapid-amplification of cDNA ends (RACE)
114
technology to amplify the ends of the fragments using a 5’-Full RACE kit (Clontech,
115
USA) and a 3’-Full RACE kit (TaKaRa). All primers are listed in Table 1, and
116
sequencing was performed as described above, followed by alignment splicing.
117
2.3. Sequence analysis
118
Sequence alignment was performed using the NCBI database and the BLAST tool
119
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). The open reading frame (ORF) of the ScGal
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gene was obtained using ORF finder (http://www.ncbi.nlm.nih.gov/gorf/orFigure.cgi).
121
Protein
122
(http://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cg). Signal peptide analysis was
123
performed
124
three-dimensional structure of ScGal was predicted using SWISS-MODEL
125
(http://swissmodel.expasy.org/). Amino acid sequences of galectins in other species
126
were obtained from GenBank. Multiple sequence alignment and phylogenetic
127
analyses were performed using the BioEdit Sequence Alignment Editor and MEGA
128
5.0 software, respectively.
129
2.4. Real-time PCR analysis of ScGal mRNA expression
130
A total of 500 ng of RNA for each sample was reverse-transcribed using a
131
PrimeScript RT reagent Kit (TaKaRa) according to the manufacturer's instructions.
132
Primer 5.0 software was used to design specific primers (Table 1) and optimisation
133
was performed by melting curve analysis. Previous studies showed that 18S rRNA is
134
a suitable housekeeping gene in S. constricta (Niu et al., 2013a, 2014). Quantitative
135
real-time PCR (qRT-PCR) was performed with 20 µl reactions containing 1.6 µl of
SignalP
predicted
by
conserved
domain
prediction
(http://www.cbs.dtu.dk/services/SignalP-2.0/).
The
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ACCEPTED MANUSCRIPT cDNA, 10 µl of 2× SYBR Premix Ex Taq, 0.8 µl of each primer, and 6.8 µl of ddH2O.
137
Thermal cycling conditions included an initial denaturation at 95°C for 3 min,
138
followed by 40 cycles at 95°C for 5 s, and 60°C for 30 s. The Fluorescent signal for
139
the dissolution curve was measured, and relative expression levels were determined
140
using the 2-∆∆Ct method.
141
2.5. Responses of ScGal following bacterial challenge
142
Clams were kept in seawater at 25°C and 20‰ salinity for 1 week, and 300 adult
143
clams (mean shell length of 5.3 ± 0.2 cm) were randomly divided into three groups of
144
100 animals each. Members of each group were injected in the foot with 50 µl of
145
Staphylococcus aureus or Vibrio anguillarum in phosphate-buffered saline (PBS;
146
2×109 cells/ml), or PBS alone as a control. Hemolymph were collected at 0, 4, 8, 12,
147
24, 48 and 72 h post-inoculation. RNA extraction, cDNA synthesis, qRT-PCR, and
148
data analysis were performed as described in section 2.4.
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To reduce experimental error, extracting nine individuals at each time point to
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extract RNA from hemolymph, equally mixed every three RNA samples for reverse
151
transcription of cDNA，and a total of three replicates for qRT-PCR. One-way
9
ACCEPTED MANUSCRIPT analysis of variance was performed using SPSS software to determine significant
153
differences between experimental and control groups. Differences were considered
154
significant at p <0.05 (*) and highly significant at p <0.01 (**).
155
2.6. Expression and purification of recombinant rScGal
156
Primers with restriction enzyme sites were designed based on both ends of the cDNA
157
fragment encoding the mature ScGal protein (Table 1). Following amplification and
158
gel purification, the fragment was digested with BamHI and XhoI (TaKaRa) at 37°C
159
for 1 h, and the expression vector pET-28a (Novagen, Germany) was also digested.
160
The digested fragment was then ligated with the vector overnight using T4 ligase
161
(TaKaRa) and the resulting recombinant plasmid (pET-28a-ScGal) or empty pET-28a
162
vector (negative control) were separately transformed into E. coli BL21 (DE3) cells.
163
Nucleic acid sequencing was performed to confirm the successful introduction of the
164
desired plasmid. Bacteria were cultured at 37°C until the absorbance at 600 nm
165
reached 0.6−0.8. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was then added at a
166
final concentration of 0.5 mM to induce protein expression, and culturing was
167
continued at 18°C for 16 h. Bacteria were collected by centrifugation at 7,500 × g for
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ACCEPTED MANUSCRIPT 5 min, washed three times with PBS, resuspended in PBS containing 1% protease
169
inhibitor, and sonicated on ice. The supernatant containing soluble protein was
170
collected by centrifugation at 10,000 × g at 4°C for 10 min, and passed through a
171
pre-equilibrated Ni2+-chelating Sepharose column (GE Healthcare, USA). The
172
His-tagged target protein was purified and eluted with 200 mM imidazole in PBS (pH
173
7.4).
174
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), passed through a Desalting
175
Gravity Column (Sangon, China), eluted in PBS, and the protein concentration was
176
determined using a bicinchroninic acid (BCA) kit (CWBIO, China).
177
2.7. Binding of rScGal to microorganisms and carbohydrates
178
Based on previous studies (Zhang et al., 2009), the binding activity of rScGal was
179
tested against Gram-positive bacteria (S. aureus, Bacillus subtilis and Streptococcus
180
agalactiae) and Gram-negative bacteria (Aeromonas hydrophila, E. coli and V.
181
anguillarum). Carbohydrates were added to inhibit this process to investigate the
182
binding of rScGal to bacterial surface glycans. Briefly, an overnight culture of
183
bacteria was centrifuged at 7,500 × g for 10 min, washed twice with PBS, then
eluted
protein
was
analysed
by
12%
sodium
dodecyl
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ACCEPTED MANUSCRIPT thoroughly resuspended in PBS to an OD600 of 1.0. Purified recombinant protein (5
185
µg) was then incubated with 500 µl of bacterial suspension with slight shaking for 20
186
min at room temperature. Bacteria were centrifuged, washed four times with PBS, and
187
eluted with 7% SDS for 1 min. Eluted proteins were resuspended in SDS-PAGE
188
loading buffer and separated by 12% SDS-PAGE, followed by western blotting
189
analysis using anti-His antibody. Subsequently,
190
lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) were
191
separately incubated with recombinant protein (5 µg) for 1 h at room temperature (50
192
µl of 1 mg/ml for each carbohydrate). In the control group, recombinant protein was
193
incubated with PBS alone. Finally, 500 µl of V. anguillarum suspension was added
194
and the binding assay was performed as described above.
195
2.8. Bacterial agglutination assay
196
Gram-positive bacteria (S. aureus, B. subtilis and S. agalactiae) and Gram-negative
197
bacteria (A. hydrophila, E. coli and V. anguillarum) were separately cultured
198
overnight, collected by centrifugation, washed three times with PBS, and resuspended
199
in 0.1 M Na2CO3 (pH 9.5) to a final concentration of 109 cells/ml. Fluorescein
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maltose,
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ACCEPTED MANUSCRIPT isothiocyanate (FITC; Solarbio, China) was added at a final concentration at 0.5
201
mg/ml and incubated for 30 min at room temperature to ensure thorough labelling.
202
After three washes to completely remove unlabelled FITC, bacteria were resuspended
203
in PBS to a density of 109 cells/ml. Next, 20 µl of rScGal (100 µg/ml) was mixed with
204
labelled bacteria and incubated for 1 h at room temperature with gentle shaking to
205
ensure mixing, after which agglutination was observed using a fluorescence
206
microscope (Leica, Germany).
207
2.9. Effect of rScGal on phagocytosis in hemocytes
208
Labelling of V. anguillarum and S. aureus with FITC was performed as described in
209
Section 2.8. Fresh hemocytes from clams were washed three times with PBS by
210
centrifugation at 1000 × g for 5 min and diluted to 1×107 cells/ml using a flow
211
cytometer. A 200 µl sample of bacterial suspension (108 cells/ml) was mixed with 180
212
µl of hemocytes suspension and 20 µl of rScGal in a 1.5ml centrifuge tube. In the
213
negative control, rScGal was replaced with bovine serine albumin (BSA) and PBS.
214
Samples were then incubated in the dark at room temperature, mixed once every 5
215
min, and analysed using a flow cytometer.
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ACCEPTED MANUSCRIPT 3. Results
217
3.1. Characterisation of the ScGal sequence
218
A galectin gene fragment was identified from the cDNA library of S. constricta (Niu
219
et al., 2013b), and the full-length cDNA sequence was obtained and designated ScGal.
220
It comprises 1066 bp with a 531 bp coding sequence (CDS) including a stop codon,
221
an 84 bp 5′ untranslated region (UTR), and a 451 bp 3′ UTR with a predicted
222
polyadenylation signal sequence (1026AATAAA1031) and a poly(A) tail. The ORF
223
encodes a protein of 176 amino acid residues without a signal peptide, with a
224
calculated molecular mass of 19.77 kDa and the theoretical isoelectric point of 5.65.
225
The sequence has only one CRD domain (residues 48−174), and this is the Gal with a
226
single CRD among known aquatic mollusc sequences (Fig. S1).
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Multiple sequence alignment of ScGal with orthologs from aquatic molluscs
228
revealed relatively high sequence conservation (Fig. S2), and some conservation with
229
vertebrates (Orycteropus afer afer) and arthropods (Pteropus alecto).
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To evaluate the molecular phylogeny of ScGal, a phylogenetic tree was
231
constructed based on the sequences of galectin orthologs from different taxa using the
14
ACCEPTED MANUSCRIPT neighbour-joining method (Fig. S3). The tree forms two distinct clusters for
233
invertebrates and vertebrates, and among invertebrates, ScGal is most closely related
234
to galectin-2 from Mytilus galloprovincialis.
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Prediction of the protein structure by SWISS-MODEL based on human galectin-4
236
(40.15% identity) showed that ScGal consists of a β-sandwich structure with two
237
anti-parallel β-sheet bundles. β‐strands F1−F5 form the convex side of the β‐sandwich
238
that is related to secretion (Fig. S4A). The concave side of the β‐sandwich comprises
239
β‐strands S1−S6 and contains the carbohydrate binding site (Fig. S4B), which is well
240
conserved amongst galectin family members (Bum-Erdene et al., 2015).
241
3.2. Real-time PCR analysis of ScGal mRNA expression in different tissues
242
Constitutive expression of ScGal was examined in different tissues of adult clams
243
(Fig. 1A). The mRNA transcript of ScGal is highly expressed in hemolymph and gill
244
(p <0.05), with the highest in hemolymph. Whereas expression in mantle, foot,
245
siphon, liver and gonad was comparatively low.
246
3.3. Expression of ScGal following bacterial challenge
247
As shown in Fig. 1B, at several hours after injection of S. aureus or V. anguillarum,
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ACCEPTED MANUSCRIPT ScGal expression was significantly enhanced compared with the control group. In the
249
S. aureus group, expression peaked at 12 h after injection (>3-fold), whereas
250
expression in the V. anguillarum group responded more slowly after injection and
251
reached a 3-fold increase at 72 h.
252
3.4. Expression and purification of rScGal
253
To characterise the biological functions of ScGal, E. coli BL21 (DE3) cells
254
harbouring the pET-28a-ScGal construct were cultured, and rScGal was mainly
255
expressed as in soluble form and accumulated in the supernatant (Fig. 2). SDS-PAGE
256
yielded a distinct band with a molecular weight of ~26 kDa, in accordance with the
257
predicted molecular mass of the fusion protein. The recombinant rScGal fusion was
258
purified by affinity chromatography, and a final concentration of ~1.0 mg/ml was
259
measured using a BCA kit.
260
3.5. Binding activity of rScGal
261
The six bacterial strains were separately incubated with rScGal, washed thoroughly to
262
remove unbound protein (Fig. 3A), eluted with 7% SDS, and the eluate was analysed
263
by western blotting. As shown in Fig. 3B, rScGal bound to all six bacterial strains,
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ACCEPTED MANUSCRIPT with no obvious differences between species. Next, we pre-incubated rScGal
265
separately with six different types of carbohydrates, then with V. anguillarum cells. As
266
shown in Fig. 3C, compared with the control group, all treatments inhibited binding
267
of recombinant protein to bacteria to some extent. PGN showed the strongest
268
inhibition, followed by
269
maltose had a slight effect.
270
3.6. Effect of rScGal on agglutination of bacteria
271
FITC-labelled bacteria exhibited green fluorescence under a fluorescence microscope
272
(Fig. 4). However, after incubation with rScGal, all six bacterial strains exhibited
273
agglutination, with V. anguillarum showing the strongest agglutination.
274
3.7. Effect of rScGal on phagocytosis in hemocytes
275
Flow cytometry was used to measure samples (5 µl), and the results were drawn on a
276
scatter plot. To exclude the influence of fluorescence intensity in the area where
277
bacteria are located, the area where remaining hemocytes are located was defined as
278
Gate A. Subsequently, fluorescence data for each experimental group were limited to
279
Gate A (Fig. 5A). In addition, in the fluorescence intensity graph, based on untreated
and LTA. By contrast, LPS,
D-mannose
and
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ACCEPTED MANUSCRIPT hemocytes, we defined phagocytic parts of cells (Fig. 5B), and the fluorescence
281
intensity was positively correlated with the number of phagocytic bacteria. Phagocytic
282
ability (PA) is expressed as the proportion of hemocytes engaged in phagocytosis in
283
all cells. All experiments were repeated in triplicate, and the results are shown in the
284
marker section of the scatter plot (Fig. 5C).
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Statistical analysis of the data showed that the PA of hemocytes was stronger
286
following rScGal treatment than in the BSA-treated and untreated groups for both S.
287
aureus and V. anguillarum (Fig. 6). One-way analysis of variance was performed
288
using SPSS software to determine significant differences (p <0.05) between
289
experimental and control groups.
290
4. Discussion
291
Galectins are a large family of evolutionarily conserved proteins that contain one or
292
more CRDs with binding specificity for β-galactoside residues. In recent years, many
293
studies have shown that galectin activity is non-Ca2+-dependent, and plays a key role
294
in biological innate immune activities, especially in invertebrates that lack adaptive
295
immunity (Schulenburg et al., 2007; Wang and Zhao, 2004). In mammals, galectins
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ACCEPTED MANUSCRIPT are divided into three types; proto, chimera, and tandem repeat’. However, in aquatic
297
molluscs, galectins are classified as 1-CRD, 2-CRD, 4-CRD, and chimeric ‘GREP’
298
proteins (Vasta et al., 2015). In the present study, the full-length cDNA of a galectin
299
was
300
carboxyl-terminal CRD, and was hence classified as 1-CRD member. Amino acid
301
residues in the dimerisation interface are highly conserved, suggesting that this
302
galectin may play a role in the crosslinking of glycoprotein receptors and subsequent
303
cellular signalling events (Vladoiu et al., 2015). ScGal lacks a typical secretory signal
304
peptide,
305
ER/Golgi-independent pathway (Cha et al., 2015; Shi et al., 2014).
from
razor
clam
(Sinonovacula
constricta)
with
only
one
it
might
be
secreted
to
the
extracellular
space
via
an
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ScGal mRNA transcripts were detected in all tested tissues (Fig. 1A), and
307
expression was highest in hemolymph, followed by gill. Invertebrate hemolymph are
308
involved in various aspects of surveillance and cellular immune responses (Johansson
309
et al., 2000), while gills are the first line of defence against aquatic microbial invasion
310
(Ellis, 2001). Therefore, hemolymph were selected for studying mRNA expression of
311
ScGal following microbial invasion. The results revealed rapid up-regulation that
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ACCEPTED MANUSCRIPT peaked at 12 h after S. aureus challenge, whereas ScGal mRNA transcripts increased
313
gradually after challenge with V. anguillarum, and reached 3-fold up-regulation at 72
314
h. This indicates that ScGal is involved in acute immune responses against certain
315
bacteria (such as S. aureus). The fact that expression was significantly up-regulated
316
with both bacteria indicates broad resistance to pathogens, implying that ScGal is an
317
important immune factor. Similarly, Gal in Argopecten irradians is widely expressed
318
in various tissues, and is highly expressed in hemolymph after challenge with V.
319
anguillarum and Pichia pastoris (Song et al., 2011), and in Eriocheir sinensis, EsGal
320
acts as an acute protein-mediated immune response (Wang et al., 2016). This
321
immunological activity in different species further demonstrates the pivotal role of
322
galectins in innate immunity in invertebrates.
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As PRRs, lectins activate immune responses by recognising pathogens through
324
their unique CRDs (Liu, 2005). In Amphioxus, the AmphiITLN-like protein exhibits
325
preferential binding to PGN of Gram-positive bacteria (Yan et al., 2012). In A.
326
irradians, AiGal binds to a variety of bacteria, resulting in varying degrees of
327
agglutination (Song et al., 2010). Similarly, in the present study, ScGal showed strong
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329
We pre-incubated rScGal with various carbohydrates prior to binding to bacteria to
330
explore their inhibitory effects. The results indicate that rScGal binds PGN most
331
strongly, followed by D-galactose and LTA, and the other tested carbohydrates bound
332
weakly. PGN is the main component of many bacterial cell walls, and PGN in the cell
333
wall of Gram-positive bacteria (G+) accounts for ~50% of the dry weight, while PGN
334
in the cell wall of Gram-negative bacteria (G-) accounts for only ~10%. LTA is a
335
specific component of Gram-positive bacteria (Dziarski, 2003; Schleifer and Kandler,
336
1973; Wicken and Knox, 1975). The ability of ScGal to bind and aggregate three
337
Gram-positive and three Gram-negative bacterial species in this study was mainly
338
attributed too binding PGN, LTA and D-galactose on the bacterial surface.
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It has been reported that lectins are key factors in phagocytosis (Sharon, 1984), a
340
process associated with the ability of lectin receptors to recognise antigens (Vogel et
341
al., 1980). To test whether ScGal affects phagocytosis in hemocytes, we tested two
342
bacterial strains to investigate the phagocytic rate of hemocytes with FITC-labelled
343
bacteria after incubation with rScGal. The phagocytic ability of blood cells was
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ACCEPTED MANUSCRIPT significantly enhanced against both strains after rScGal was added. Thus, ScGal not
345
only acts as a PRR in the process of immune recognition, but also participates in the
346
pathogen clearance process.
347
5. Conclusion
348
A full-length galectin (ScGal) was identified in S. constricta. ScGal was highly
349
expressed in hemolymph, and strongly up-regulated after bacterial challenge.
350
Recombinant rScGal protein activated the immune response, agglutinated bacteria,
351
and stimulated the phagocytic action of hemocytes to enhance antigen clearance.
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Acknowledgements
354
This work was supported by the National Natural Science Foundation of China (grant
355
number 31472278), the National High Technology Research and Development
356
Program of China (863 Program; grant number 2012AA10A400-3), and the Shanghai
357
Universities Knowledge Service Platform (grant number ZF1206).
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Competing interests
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ACCEPTED MANUSCRIPT 360
The authors have no competing interests to declare.
361
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Tables
Table 1 Primers used to study the ScGal gene Sequence (5′-3′)
Comment
ScGal-F1
TTATTACCCGACAGACACTTAGC
Amplification of an
ScGal-R1
TGTGGAGGTTACAGTGTGGTG
ScGal-R2
TTCCGGTCGTGGGTCTGGAGTTTG
UPM
CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACG
Primer
ScGal gene fragment 5′RACE (ScGal)
SC
CAGAGT
RI PT
cDNA cloning
CGAAGGGCACATGCGGATTGAAGG
UPM
CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACG
M AN U
ScGal-R3
CAGAGT
CACAAGGGACAAGAGAAACGCACA
OUTER
TACCGTCGTTCCACTAGTGATTT
ScGal-F3
GTTATGAAGTCGGGTGTTTGCTATGG
INNER
CGCGGATCCTCCACTAGTGATTTCACTATAGG
qPCR analysis
TE D
ScGal-F2
CACGACCGGAAATCATTGCT
ScGal-R4
CATGCGGATTGAAGGTGGTT
18S-F
TCGGTTCTATTGCGTTGGTTTT
18S-R
CAGTTGGCATCGTTTATGGTCA
expressiona ScGal-F5 ScGal-R5 a
AC C
Protein
3′RACE (ScGal)
3′RACE (ScGal)
RT-PCR (ScGal)
RT-PCR (Control)
EP
ScGal-F4
5′RACE (ScGal)
CGCGGATCCGCGATGTCAAGTAACCAAACTCCAGA
Construction
CCGCTCGAGCGGGTACGCCACCTTCGTCAGT
vectors
BamHI and XhoI sites are underlined. 492 493
32
of
ACCEPTED MANUSCRIPT Figure legends
495
Fig. 1. (A) Expression of ScGal mRNA in seven adult tissues determined by
496
qRT-PCR. Expression levels are relative to those in gonad tissue. Results are
497
representative of three independent experiments. Bars represent the mean ± standard
498
error (SE; n = 3) for each tissue. An asterisk above bars denotes a significant
499
difference (p <0.01) between tissues. (B) Expression profiles of ScGal mRNA in
500
hemocytes following challenge with Vibrio anguillarum or Staphylococcus aureus.
501
Clams injected with phosphate-buffered saline (PBS) were used as controls.
502
Differences between PBS and pathogen infection groups are shown (*p <0.05) and
503
(**p <0.01).
SC
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504
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Fig. 2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
506
analysis of the rScGal protein. Lane M, markers (25 kDa); lane 1, supernatant of
507
Escherichia coli cells harbouring the pET28a-ScGal construct after ultrasonication.
508
lane 2, purified rScGal protein.
AC C
505
509
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511
was incubated with S. aureus cells in PBS at room temperature for 20 min. After
512
centrifugation to separate unbound protein and washing four times with PBS, bound
513
protein was eluted with 7% SDS for 1 min. Recombinant ScGal was detected using
514
antiserum against ScGal. Lane 1, wash solution from S. aureus cells (PBS controls);
515
lane 2, unbound protein; lanes 3−6, wash solutions from S. aureus cells incubated
516
with ScGal; lane 7, protein eluted with 7% SDS. (B) Binding of recombinant ScGal to
517
microorganisms. Recombinant ScGal was incubated with microorganisms as
518
described in panel A, and protein eluted with 7% SDS was detected by
519
immunoblotting with antiserum against ScGal. (C) Inhibition of the bacterial binding
520
activity of rScGal by carbohydrates. Different carbohydrates were added to test
521
whether they could inhibit binding to bacterial cells.
SC
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522
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510
523
Fig. 4. Agglutinating activity of rScGal against FITC-labelled V. anguillarum, E. coli,
524
Aeromonas hydrophila, S. aureus, Streptococcus agalactiae and Bacillus subtilis. PBS
525
was incubated with bacteria as a negative control.
34
ACCEPTED MANUSCRIPT 526
Fig. 5. Flow cytometry analysis of phagocytosis by hemocytes following exposure to
528
ScGal-treated, BSA-treated, or PBS-treated V. anguillarum or S. aureus. (A) Scatter
529
plots for V. anguillarum alone (left panel) and V. anguillarum + hemocytes. The area
530
where hemocytes are located is defined as Gate A. (B) Graph showing the
531
fluorescence intensity of hemocytes alone, revealing parts involved in phagocytosis.
532
(C) Phagocytosis in hemocytes exposed to PBS-, BSA- or ScGal-treated bacteria.
533
Results are averages of three independent experiments.
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Fig. 6. Analysis of phagocytosis after treatment with rScGal (or a negative control).
536
Differences between groups are shown (*p <0.05; **p <0.01).
AC C
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ACCEPTED MANUSCRIPT Figures
539
Fig.1.
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540
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541
36
ACCEPTED MANUSCRIPT Fig.2.
SC
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542
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ACCEPTED MANUSCRIPT Fig.3.
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545
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546
38
ACCEPTED MANUSCRIPT Fig.4.
AC C
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547
548 549
39
ACCEPTED MANUSCRIPT Fig.5.
EP
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AC C
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TE D
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550
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ACCEPTED MANUSCRIPT Fig.6.
EP
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ACCEPTED MANUSCRIPT 556 557
Supplementary data
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Fig. S1. Detection of putative conserved domains of Sinonovacula constricta ScGal
560
using the NCBI conserved domain database (CDD). The C-terminus (residues
561
48−174) is a carbohydrate recognition domain (CRD). Residues in the dimerisation
562
interface and sugar binding pocket are indicated by triangles.
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563
42
564
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Fig. S2. Multiple sequence alignment ScGal from Sinonovacula constricta and related
566
proteins from various species using sequences obtained from the NCBI database.
567
Conserved amino acid residues are shaded dark grey, and similar amino acids are
568
shaded light grey. Residues involved in the dimerisation interface are in red boxes,
569
and the CRD is in a black box.
AC C
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Fig. S3. Phylogenetic tree showing homology between ScGal and galectins from
572
various vertebrates and invertebrates. The tree was constructed using a
573
ClustalW-generated multiple sequence alignment of amino acid sequences using the
574
neighbour-joining method in MEGA 5. The topological stability of trees was
575
evaluated by 10,000 bootstrapping replications. Numbers at branches indicate
576
bootstrap values (%).
AC C
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Fig. S4. The predicted 3D structure of ScGal. The protein fold consists of a
579
β-sandwich with two anti-parallel β-sheet bundles. (A) β‐strands F1−F5 form the
580
convex side of the β‐sandwich. (B) The concave side of the β‐sandwich comprises
581
β‐strands S1−S6. A bound lactose molecule in shown stick representation (grey
582
carbons and blue oxygens).
AC C
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45
ACCEPTED MANUSCRIPT Highlights
- A single CRD galectin was identified in razor clam (Sinonovacula constricta)
RI PT
- ScGal mRNA is mainly expressed in hemolymph and gill
- ScGal expression is up-regulated following bacterial challenge
SC
- ScGal aggregates bacteria, and binds PGN, LTA and D-galactose
AC C
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- ScGal also stimulates hemocytes to phagocytose invading bacterial pathogens