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Characterisation of Scavenger Receptor Class B Type 1 in Rare Minnow (Gobiocypris Rarus) T

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Characterisation of Scavenger Receptor Class B Type 1 in Rare Minnow (Gobiocypris Rarus) T Fish and Shellfish Immunology 89 (2019) 614–622 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article Characterisation of scavenger receptor class B type 1 in rare minnow (Gobiocypris rarus) T ∗∗ ∗ Mi Oua, Rong Huangb, Qing Luoa, Lv Xiongb, Kunci Chena, , Yaping Wangb, a Key Laboratory of Tropical and Subtropical Fishery Resources Application and Cultivation, Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510380, China b State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China ARTICLE INFO ABSTRACT Keywords: Scavenger receptor class B type 1 (SRB1) is a transmembrane protein belonging to the scavenger receptors (SRs) Rare minnow family and it plays an important role in viral entry. Not much is known on SRB1 in teleost fish. Grass carp GCRV reovirus (GCRV) cause huge economic losses in grass carp industry. In this study, rare minnow (Gobiocypris SRB1 rarus) was used as a model fish to investigate the mechanism of GCRV infection, which is sensitive to GCRV. The Receptor structure of SRB1 gene in G. rarus (GrSRB1) was cloned and elucidated. GrSRB1 is composed of 13 exons and 12 Cell entry introns, and its full-length cDNA is 2296 bp in length, with 1521 bp open reading frame (ORF) that encodes a 506 amino acid protein. The GrSRB1 protein is predicted to contain a typical CD36 domain and two trans- membrane regions. In G. rarus, GrSRB1 is expressed strongly in the liver (L), intestines (I), brain (B) and muscle (M), while it is expressed poorly in the heart (H), middle kidney (MK), head kidney (HK) and gills (G). After infection with GCRV, GrSRB1 expression was up-regulated in main immune tissues during the early infection period. Moreover, co-immunoprecipitation assays revealed that GrSRB1 could interact with the outer capsid protein of GCRV (VP5 and VP7). These results suggest that GrSRB1 could be a receptor for GCRV. We have managed to characterize the GrSRB1 gene and provide evidence for its potential functions for GCRV entry into host cells. 1. Introduction with multiple glycosylation sites and two short intracellular tails [8]. SR-BI is a physiologically relevant HDL receptor that mediates selective Scavenger receptors (SRs) are a group of endocytic pattern re- uptake of lipoprotein (HDL)-derived cholesteryl ester (CE) in vitro and cognition receptors (PRRs) with ligand-binding activity, which directly in vivo [9,10]. SR-BI facilitates the bidirectional flux of free cholesterol bind to proteins, polyribonucleotides, polysaccharides and lipids [1,2]. (FC) and phospholipids between HDL and cells, thus influencing plasma SRs constitute a large family of proteins that are structurally diverse membrane cholesterol content [11,12]. It has also been implicated in and participate in a wide range of biological functions, such as fatty the entry into cells of the hepatitis C virus (HCV) [13–15], protection acid metabolism, innate immunity, endocytosis of modified lipopro- against female infertility [16], phagocytosis of apoptotic cells [17], teins, phagocytic clearance of unusable components, like modified host plasmodium infection [18,19], chlamydia trachomatis survival in host molecules and apoptotic cells, and also pheromone signaling [3–5]. cells [20], and plays a regulatory role in HDL induced signaling in the Based on their protein domain architecture and biological function, SRs vasculature [21,22]. Recent studies of SR-BI showed protection against have been discovered and the definition has been broadened to eight septic death likely through its role in modulating immune response via different classes (A-H) in mammals [6]. TLR4 signaling in macrophages and through its role in removal of LPS Scavenger receptors class B (SR-Bs) include scavenger receptor class from circulation [23]. SR-B1 and LOX-1 participate in BEC activation B type I (SR-BI) (alternative names: SR-B1, SCARB1), SR-BII, cluster triggered by the TLR3 ligand dsRNA in vitro, and regulate the recruit- determinant 36 (CD36) and lysosomal integral membrane protein 2 ment of inflammatory cells into the lungs and the activation of T cells (LIMP2) in mammals [7]. All SR-Bs are type III transmembrane re- and dendritic cells in draining lymph nodes [24]. Furthermore, SR-B1 ceptors with two transmembrane (TM) domains, an extracellular loop enhances bacterial adhesion and clinical signs of systemic inflammatory ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (K. Chen), [email protected] (Y. Wang). https://doi.org/10.1016/j.fsi.2019.04.031 Received 25 January 2019; Received in revised form 27 March 2019; Accepted 10 April 2019 Available online 13 April 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved. M. Ou, et al. Fish and Shellfish Immunology 89 (2019) 614–622 response syndrome in pyometra-affected uteri [25]. 2.2. GCRV challenge and sampling SR-BI has been described in many species such as human, mouse [26], shrimp [27,28], and crab [5,29,30], but functional studies on Before GCRV challenge, five healthy fish were sacrificed as one bony fish SR-BI proteins are limited. Kleveland et al. [31] reported that group, and samples from the gills (G), liver (L), spleen (S), intestine (I), SR-BI has an important function in intestinal lipid absorption in Atlantic middle kidney (MK), muscle (M), head kidney (HK), heart (H), and salmo (Salmo salar L.). Raldúa and Babin [32] pointed out that BLT-1 brain (B) were collected. At the same time, fin tissue was obtained and inhibits SR-BI activity and induces a copper-dependent phenotype fixed in 95% ethanol. GCRV (GD108 strain) was used for challenge during zebrafish (Danio rerio) development. Sundvold et al. [33] found experiments, and the operation was referring to Ou et al. reported [38]. a novel paralog of SCARB1 (SCARB1-2) in Atlantic salmon, and thought Five individuals were killed and tissues including gills (G), liver (L), this gene may be a possible source of genetic variation in salmonid flesh spleen (S) and intestine (I) were collected at 0, 6, 12, 18, 24, 48, 72, 96, pigmentation. Eslamloo et al. [34] indicated that there is a time-de- 120, and 144 h after GCRV exposure. Three groups we collected at each pendent down-regulation for scarb1-a, scarb1-b and csf1r in salmon time points. All samples were immediately homogenized in TRIzol re- macrophage-like cells (MLCs) within the polyriboinosinic poly- agent (Invitrogen, USA) and stored at −80 °C until RNA extraction. ribocytidylic acid (pIC) stimulation at 24 h post-stimulation compared to the earlier time point. Because of the multiple function of SR-B1, it is necessary to conduct studies in bony fishes. 2.3. RNA extraction and cDNA synthesis Grass carp (Ctenopharyngodon idellus) is a crucial aquaculture spe- cies in China, but tremendous economic loss is often caused by Grass The total RNAs were extracted according to the manufacturer's in- Carp Reovirus (GCRV), a dsRNA virus. The genome of GCRV contains struction for TRIzol reagent. Using random hexamer primer (Takara, 11 segments of linear dsRNA, which encodes seven structural proteins Japan) and M-MLV Reverse Transcriptase (Promega, USA), first strand (VP1-VP7) and five nonstructural proteins (NS80, NS38, NS31, NS26, cDNA synthesis which used for the fragment amplification were per- and NS16), respectively. VP1-VP4 and VP6 are the components of the formed on liver-derived RNA; SMART™ RACE cDNA Amplification Kit viral core, and the outer GCRV capsid is composed of 200 trimers of (Takara, Japan) was used to obtain 5′-RACE Ready cDNA and 3′-RACE VP5 and VP7 heterodimers [35]. It is essential to investigate the pa- Ready cDNA; ReverTra Ace qPCR RT Kit(Toyobo, Japan)was utilized to thogenesis of GCRV infection and look for preventative measures to synthesize cDNA that were used as templates for qRT-PCR. promote sustainable culture of grass carp. However, the bigger size and longer reproductive cycle of the grass carp make it difficult to study the mechanism. The rare minnow (Gobiocypris rarus), which is a small cy- 2.4. Full-length cDNA cloning and sequence analysis for GrSRB1 prinid species, has been recognized as a useful model for aquatic toxi- city testing, chemical safety assessments, and antiviral breeding [36]. To identify GrSRB1 cDNA sequences, primers GrSRB1-F1 and Also, it is very sensitive to GCRV, and its mortality is 100%. GrSRB1-R1 (Table 1) were designed and synthesized based on the In our previous study, transcriptome of GCRV-infected C. idellus highly conserved regions of known fish SRB1 sequences including D. kidney (CIK) cells showed that srb1, itgb2 and sec22b appear to be up- rerio (DrSRB1, Accession no. NM_198921.2), Oryzias latipes (OlSRB1, regulated during 8 –24 h post infection period, which suggest that itgb2 Accession no. XM_011481416.2), Cyprinus carpio (CcSRB1, Accession and/or srb1 may be important for cell entry of GCRV [37]. In the cur- no. XM_019104218.1), Salmo salar (SsSRB1, Accession no. rent study, we employed the rare minnow as a model fish to study the NM_001123612.1), and Esox lucius (ElSRB1, Accession no. mechanism of GCRV infection. A full-length cDNA sequence encoding XM_010878228.3). The 3′ untranslated regions (UTRs) were obtained SRB1 protein from G. rarus was identified and designated as GrSRB1. according to the manufacturer's instruction for SMART™ RACE cDNA The expression patterns of GrSRB1 gene in different healthy tissues Amplification Kit. The cDNA sequence was confirmed by sequencing were analyzed, and the response to GCRV infection in immune tissues the PCR product amplified by primers GrSRB1-F1 and GrSRB1-R2 was studied. In addition, the relationship between some major outer (Table 1) within the 3′ UTRs. capsid proteins of GCRV and GrSRB1 was also investigated.
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