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Cell Surface Transglutaminase Required for Nodavirus Entry Into Freshwater Prawn Hemocytes T Fish and Shellfish Immunology 89 (2019) 108–116 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article Cell surface transglutaminase required for nodavirus entry into freshwater prawn hemocytes T Ratchanok Sirikharina,b,c,1, Tanatchaporn Utairungseea,b,c,1, Jiraporn Srisalaa, ∗ Sittiruk Roytrakuld, Siripong Thitamadeeb,c, Kallaya Sritunyalucksanaa, a Aquatic Animal Health Research Team, Integrative Aquaculture Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi Office, Rama VI Rd., Bangkok, 10400, Thailand b Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand c Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand d Proteomics Research Laboratory, Genomic Institute, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Klong Luang, Pathumthani, 12120, Thailand ARTICLE INFO ABSTRACT Keywords: To identify molecules involved in Macrobrachium rosenbergii nodavirus (MrNV) entry into hemocytes of the giant Transglutaminase freshwater prawn M. rosenbergii, biotinylated prawn hemocyte membrane proteins were prepared, purified and Macrobrachium rosenbergii separated by SDS-PAGE. The proteins were blotted on the nitrocellulose membrane before incubation with the M. rosenbergii nodavirus (MrNV) MrNV capsid protein (MrNV-CP) by a VOPBA technique. Subsequent mass spectrometry and analysis of immune- White tail disease (WTD) reactive bands represent putative binding partners including transglutaminase (TG), actin, α2-macroglobulin, Viral entry α1-tubulin, F1-ATP synthase β-subunit and a currently uncharacterized protein. The sequence of TG has been characterized and found 5 amino acids differences to a previously reported MrTG (ADX99580), mainly at its N- terminal part and thus, we named it MrTGII (KM008611). Recombinant MrTGII was prepared to produce a polyclonal antibody against it, which was successfully revealed the presence of MrTGII (100 kDa) in prawn hemocyte lysates. Using the pentylamine-biotin incorporation assay, an acyl transfer reaction was observed when hemocyte lysates were added to solutions containing MrNV-CP, suggesting that hemocyte MrTG could use MrNV-CP as the substrate. The expression levels of MrTGII were changed during the course of MrNV infection. By using immunostaining technique, location of MrTGII on the hemocyte surface was confirmed. Specific interac- tion between MrTGII with MrNV-CP in a dose-dependent manner was confirmed by in vitro ELISA assay. The highest binding activity of MrNV-CP was found with the N-terminal portion of the protein. In vitro neutralization using anti-MrTGII antibody resulted in inhibition of MrNV attachment to the hemocyte surface, accompanied by a dramatic reduction in viral replication. This is the first time that crustacean TG has been shown to be involved in viral entry, in addition to its roles in blood clotting and haematopoiesis. 1. Introduction control MrNV outbreaks in prawn culture. Transglutaminase (TG) (EC 2.3.2.13) comprises a family of Ca2+- Successful crustacean aquaculture is limited by viral disease out- dependent enzymes responsible for post-translational modification/ breaks. White tail disease (WTD) in the giant freshwater prawn cross-linking of proteins [4]. In mammals, TGs catalyze reactions for Macrobrachium rosenbergii is caused by M. rosenbergii nodavirus (MrNV), several protein substrates and are involved in intra- and extracellular usually in association with extra small virus (XSV) [1]. Affected prawns physiological functions, including cell death, cell adhesion and migra- exhibit white, opaque muscles in the abdominal segments, commonly tion, cell-matrix interactions, and stabilization and maintenance tissue accompanied by progressive reduction in feeding and swimming. integrity [5]. In crustaceans, TG is a crucial molecule in the blood Mortalities may reach between 50 and 70% within 2 weeks after (hemolymph) coagulation reaction [6] which is part of the innate im- transfer of post-larvae (PL) to grow-out ponds, causing high economic mune defense mechanism. It was first reported as a key coagulation losses [2,3]. Until now, there is no strategy other than prevention to factor in the system of the freshwater crayfish Pacifastacus leniusculus ∗ Corresponding author. E-mail address: [email protected] (K. Sritunyalucksana). 1 Two authors have equally contribution to the work. https://doi.org/10.1016/j.fsi.2019.03.052 Received 15 October 2018; Received in revised form 6 February 2019; Accepted 24 March 2019 Available online 27 March 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved. R. Sirikharin, et al. Fish and Shellfish Immunology 89 (2019) 108–116 [7]. Later, crayfish TG was also shown to be involved in hemocyte centrifugation at 3000×g for 10 min at 4 °C and soluble biotinylated homeostasis since loss of TG activity in hematopoietic tissue (hpt) hemocyte surface proteins were purified by streptavidin affinity beads caused the stem cells to differentiate and move into the peripheral (GE healthcare, USA) according the manufacturer's protocol. The eluted circulation [8]. In the black tiger shrimp Penaeus (Penaeus) monodon, fraction was collected and called cell surface proteins (CSPs). Acetone two types of TGs called PmTGI [9] and PmTGII [10] have been re- precipitation was used to concentrate the CSPs. ported. PmTGII and its heterologously expressed form (rPmTGII) could catalyze stable clot formation [10] but PmTGI (first reported from a 2.3. Virus overlay protein binding assay (VOPBA) hemocyte cDNA library) could not [9]. The TG of Pacific white shrimp, P. (Litopenaeus) vannamei could also be divided into two types based on To identify hemocyte cell surface proteins that interact with rMrNV- sequence comparison and phylogenetic analysis [11]. The sequence of CP, VOPBA was carried out using CSPs (30 μg) that had been previously TG from M. rosenbergii (MrTG) was found to be most closely related to separated on 10% SDS-PAGE and transferred to nitrocellulose mem- that of shrimp PmTGII [12]. MrTG-mRNAs were found in various tissues branes (GE Healthcare, USA). The blotted membranes were treated with but mainly in hemocytes. After injection with bacterial cells of Lacto- 5% skim milk in TBS buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% coccus garvieae, MrTG expression and activity was depressed and this tween-20, pH 7.4) at room temperature for 1 h with constant agitation. resulted in a delay in hemolymph clotting time [12]. The mRNA re- After washing twice, the blot was incubated with 0.5 mg of purified lative expression level of MrTG was also significantly changed after rMrNV-CP in TBS dilution buffer (TBS buffer containing 0.02% BSA) at challenges with Vibrio harveyi or with white spot syndrome virus 4 °C, overnight. To detect the rMrNV-CP binding protein(s), the mem- (WSSV) [13]. In P. monodon, infection with Taura syndrome virus (TSV) brane blot were thoroughly washed with TBS buffer before being in- was accompanied by poor hemolymph coagulation as a result of de- cubated with 1:500 dilution of anti-MrNV-CP antibody at 4 °C, over- creased TG activity [14]. In Chinese shrimp, P. (Fenneropenaeus) chi- night. After washing, the blots were incubated with alkaline nensis, WSSV was reported to affect TG expression in shrimp hemocytes phosphatase-conjugated goat anti-mouse IgG antibody (Zymed, USA) at [15]. a dilution of 1:1,000 in TBS dilution buffer at 4 °C for 4 h. Positive In this study, we found that TG in M. rosenbergii not only involved in signals were visualized by staining the blot with a commercial solution blood coagulation but also required for MrNV entry into hemocytes. of the alkaline phosphatase substrate NBT/BCIP (Invitrogen, USA). The MrTGII has been shown to bind to the capsid protein of MrNV (MrNV- NBT/BCIP reaction was stopped by extensive washing with dH2O. CP) by VOPBA (Virus Overlay Protein Binding Assay) analysis. MrTGII- cDNA sequence has been characterized and cloned into the bacterial 2.4. Mass spectrometry analysis expression vector. The recombinant MrTGII and the antibodies raised against each region of MrTGII were synthesized to confirm its function Candidate cell surface binding proteins to rMrNV-CP were excised involved in viral entry. By using immunofluorescence microscopy, it from gels stained with coomassie blue. In-gel digestion was performed was found that prawn MrTGII was localized on the surface of adult overnight with trypsin at 37 °C before carrying out Liquid chromato- prawn hemocytes and that pre-incubation of the hemocytes with anti- graphy-electrospray ionization tandem mass spectrometry (LC-MS/MS). MrTGII antibody inhibited viral replication. Specific binding between Results from LC-MS/MS were analysed by the MASCOT program MrTGII and MrNV-CP was also shown using an in vitro binding assay. (http://www.matrixscience.html). Sequence homology searches were Finally, using the pentylamine-biotin incorporation assay, MrNV-CP performed using the BLAST program with the NCBI database (http:// might be a substrate for MrTGII. Thus, all the results indicated that blast.ncbi.nlm.nih.gov.html). MrTGII has a novel functional role in viral binding and entry into giant freshwater prawn hemocytes. 2.5. The pentylamine-biotin incorporation assay 2. Materials and methods This assay
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