Molecular cloning and cellular localization of the scavenger receptor SCARF1 in common carp

TY - JOUR

T1 - Molecular cloning and cellular localization of the scavenger receptor SCARF1 in common carp

AU - Østergaard, A.E.

AU - Fink, I.R.

AU - Sukinta, A.

AU - Yixian, L.

AU - Forlenza, M.

AU - Wiegertjes, G.F.

PY - 2013

Y1 - 2013

N2 - The recognition of pathogens by the innate immune system relies on a wide range of germ-line encoded pattern recognition receptors of which some belong to the large superfamily of scavenger receptors (SRs). These receptors are expressed on cells surveying potential portals of entry, including macrophages, dendritic cells and endothelial cells and are involved in recognition of both endogenous and pathogen-derived ligands. Here we describe for the first time an evolutionarily conserved member of the SR family, named scavenger receptor class F-member1 (SCARF1), in the common carp (Cyprinus carpio). Orthologs of mammalian SCARF1 have been reported in nematodes and shown to be involved in recognition of e.g. beta-glucans. Also, SCARF1 has been shown to mediate an inflammatory cytokine response upon ligand binding whilst functioning as co-receptor for Toll-like receptors (TLRs). We found two SCARF1 genes in the common carp genome, confirming the hypothesis that carp has undergone an extra genome duplication event compared to zebrafish, where only one SCARF1 gene is found. The two SCARF1 genes in carp are 94% similar and show comparable mRNA expression levels. Sequence analysis of SCARF1 reveals a high level of amino acid conservation compared to the human orthologue. Phylogenetic analysis of carp SCARF1 with other vertebrate SCARF1 genes groups carp and zebrafish together with high bootstrap values. Synteny studies show conserved linkage with several neighbouring genes when comparing genomic regions from different species. Gene expression analysis of SCARF1 shows expression in several organs, whereas gene expression analysis of purified cell populations shows highest expression in endothelial cells, but also macrophages, granulocytes, thrombocytes and thymocytes show SCARF1 gene expression. In contrast, no expression was observed in B cells. Previously, our group has cloned TLR2 from common carp and we are now investigating the possible role of SCARF1 acting as an internalizing receptor that would facilitate TLR activation upon phagocytosis of ligands. The localization of SCARF1 was studied by confocal microscopy of human (HEK293) and fish (EPC) cell lines transfected with a fluorescently tagged receptor. In addition, antibodies directed against a V5-tagged version of SCARF1 were also used to detect SCARF1 localization. These studies showed that carp SCARF1 is expressed in the cytoplasm, but also on the cell membrane, which is comparable to human SCARF1 localization. We are performing phagocytosis studies in order to verify the internalizing ability of SCARF1 and its potential role in TLR signaling.

AB - The recognition of pathogens by the innate immune system relies on a wide range of germ-line encoded pattern recognition receptors of which some belong to the large superfamily of scavenger receptors (SRs). These receptors are expressed on cells surveying potential portals of entry, including macrophages, dendritic cells and endothelial cells and are involved in recognition of both endogenous and pathogen-derived ligands. Here we describe for the first time an evolutionarily conserved member of the SR family, named scavenger receptor class F-member1 (SCARF1), in the common carp (Cyprinus carpio). Orthologs of mammalian SCARF1 have been reported in nematodes and shown to be involved in recognition of e.g. beta-glucans. Also, SCARF1 has been shown to mediate an inflammatory cytokine response upon ligand binding whilst functioning as co-receptor for Toll-like receptors (TLRs). We found two SCARF1 genes in the common carp genome, confirming the hypothesis that carp has undergone an extra genome duplication event compared to zebrafish, where only one SCARF1 gene is found. The two SCARF1 genes in carp are 94% similar and show comparable mRNA expression levels. Sequence analysis of SCARF1 reveals a high level of amino acid conservation compared to the human orthologue. Phylogenetic analysis of carp SCARF1 with other vertebrate SCARF1 genes groups carp and zebrafish together with high bootstrap values. Synteny studies show conserved linkage with several neighbouring genes when comparing genomic regions from different species. Gene expression analysis of SCARF1 shows expression in several organs, whereas gene expression analysis of purified cell populations shows highest expression in endothelial cells, but also macrophages, granulocytes, thrombocytes and thymocytes show SCARF1 gene expression. In contrast, no expression was observed in B cells. Previously, our group has cloned TLR2 from common carp and we are now investigating the possible role of SCARF1 acting as an internalizing receptor that would facilitate TLR activation upon phagocytosis of ligands. The localization of SCARF1 was studied by confocal microscopy of human (HEK293) and fish (EPC) cell lines transfected with a fluorescently tagged receptor. In addition, antibodies directed against a V5-tagged version of SCARF1 were also used to detect SCARF1 localization. These studies showed that carp SCARF1 is expressed in the cytoplasm, but also on the cell membrane, which is comparable to human SCARF1 localization. We are performing phagocytosis studies in order to verify the internalizing ability of SCARF1 and its potential role in TLR signaling.

U2 - 10.1016/j.fsi.2013.03.278

DO - 10.1016/j.fsi.2013.03.278

M3 - Meeting Abstract

VL - 34

SP - 1727

EP - 1727

JO - Fish and Shellfish Immunology

T2 - Fish and Shellfish Immunology

JF - Fish and Shellfish Immunology

SN - 1050-4648

IS - 6

ER -