Scavenger Receptor in Fish Is a Lipopolysaccharide Recognition Molecule Involved in Negative Regulation of NF-κB Activation by Competing with TNF Receptor-Associated Factor 2 Recruitment into the TNF-α Signaling Pathway

Abstract

Scavenger receptors (SRs) play crucial roles in innate immunity by acting as pattern recognition receptors. Although SRs are widely documented in mammals, data on their occurrence and functions in ancient vertebrates are limited. In this study, we report, to our knowledge, the first cloning and functional characterization of an SR molecule from teleost fish (Tetraodon nigroviridis). This SR (TnSR) was identified as a homolog to mammalian scavenger receptor class A member 5 with the conserved structure of a class A SR. TnSR contained multidomains in a type II transmembrane receptor, including an SR cysteine-rich domain, two coiled-coil collagenous domains, a transmmebrane domain, and a short N-terminal intracellular region with an unexpected TNFR-associated factor 2-binding consensus motif similar to that in human MSR molecules. Phylogenetic analysis suggested that TnSR may be an ancient member of class A SRs resulting from the close relationship between scavenger receptor class A member 5 and macrophage SR in vertebrates associated with the subtle differences in TnSR structure. Subcellular localization analysis showed that TnSR was a cell membrane receptor with homotrimer forms involved in the recognition and internalization of LPS from surface membranes into lysosomes. Functionally, TnSR expression was dramatically induced by LPS stimulation. TnSR served as a negative regulator in LPS-induced NF-κB activation by the competitive recruitment of TNFR-associated factor 2 from the TNF-α signaling pathway. To our knowledge, this is the first report showing that SR plays an inhibitory role in LPS-elicited inflammation by cross-talking with the TNF-α inflammatory pathway. These findings contribute to a better understanding of the biological and evolutionary history of the SR family.

Class-A scavenger receptor function and expression in the rainbow trout (Oncorhynchus mykiss) epithelial cell lines RTgutGC and RTgill-W1

“…SR-A sequences are beginning to be identified in teleost fish. A SCARA4 (CL-P1) homolog has been cloned in zebrafish, Danio rerio, [11] and a SCARA5 homolog has been cloned in puffer fish, Tetraodon nigroviridis [12]. Zebrafish SCARA4 binds both mLDLs and bacteria and is involved in vasculogenesis [11], while puffer fish SCARA5 is able to bind LPS and negatively affect the pro-inflammatory response [12].…”

Essential Roles of TIM-1 and TIM-4 Homologs in Adaptive Humoral Immunity in a Zebrafish Model

“…The ORFs of DrTIM-1 and DrTIM-4 were inserted into pEGFP-N1 (Clontech), pCMV-Tag2B (Stratagene), and pcDNA6/myc-His B (Invitrogen) to construct eukaryotic expression vectors (pEGFP-DrTIM-1, pEGFP-DrTIM-4, pCMV-DrTIM-1, and pcDNA6-DrTIM-4) with enhanced GFP, flag-tag, and myc-tag, respectively (42). The primers used in plasmid constructions are shown in Supplemental Table I.…”

Characterization of SIGIRR/IL-1R8 Homolog from Zebrafish Provides New Insights into Its Inhibitory Role in Hepatic Inflammation

“…Real-time PCR was performed using a Mastercycler ep realplex instrument (Eppendorf) with a SYBR Premix Ex Taq kit (Takara Bio), following the manufacturers' instructions as previously described (31,32). Briefly, the reaction mixtures in a total volume of 10 ml were incubated for 2 min at 95˚C, followed by 40 cycles of 15 s at 95˚C, 15 s at 60˚C, and 20 s at 68˚C.…”

“…These cells were transfected with pEGFP-SIGIRR plasmid DNA (1-2 mg) using FuGENE HD transfection reagent (Promega) in accordance with the manufacturer's instructions (31,32). At 24 h posttransfection, the cells were fixed in 4% paraformaldehyde for 10 min and stained with 10 mM DiI (Beyotime) at 37˚C for 5 min.…”

“…The empty plasmid was added to ensure that all samples received equal amounts of DNA. At 24 h after microinjection, embryos were collected for luciferase activity detection as previously described (32,33). For IFN production assay, plasmids in various combinations (pEGFP-TRIF alone, pEGFP-TRIF plus DrMO, and pEGFP-TRIF plus DrMO and DrSIGIRR mRNA) were microinjected into one-cell stage embryos.…”