PDF(Pubmed)
Abstract：
The basal chordate amphioxus is a model for tracing the origin and evolution of vertebrate immunity. To explore the evolution of immunoreceptor signaling pathways, we searched the associated receptors of the amphioxus Branchiostoma belcheri (Bb) homolog of immunoreceptor signaling adaptor protein Grb2. Mass-spectrum analysis of BbGrb2 immunoprecipitates from B. belcheri intestine lysates revealed a folate receptor (FR) domain- and leucine-rich repeat (LRR)-containing protein (FrLRR). Sequence and structural analysis showed that FrLRR is a membrane protein with a predicted curved solenoid structure. The N-terminal Fr domain contains very few folate-binding sites; the following LRR region is a Slit2-type LRR, and a GPI-anchored site was predicted at the C-terminus. RT-PCR analysis showed FrLRR is a transcription-mediated fusion gene of BbFR-like and BbSlit2-N-like genes. Genomic DNA structure analysis implied the B. belcheri FrLRR gene locus and the corresponding locus in Branchiostoma floridae might be generated by exon shuffling of a Slit2-N-like gene into an FR gene. RT-qPCR, immunostaining, and immunoblot results showed that FrLRR was primarily distributed in B. belcheri intestinal tissue. We further demonstrated that FrLRR localized to the cell membrane and lysosomes. Functionally, FrLRR mediated and promoted bacteria-binding and phagocytosis, and FrLRR antibody blocking or Grb2 knockdown inhibited FrLRR-mediated phagocytosis. Interestingly, we found that human Slit2-N (hSlit2-N) also mediated direct bacteria-binding and phagocytosis which was inhibited by Slit2-N antibody blocking or Grb2 knockdown. Together, these results indicate FrLRR and hSlit2-N may function as phagocytotic-receptors to promote phagocytosis through Grb2, implying the Slit2-N-type-LRR-containing proteins play a role in bacterial binding and elimination.
摘要：
基底脊索文昌鱼是追踪脊椎动物免疫起源和进化的模型。探索免疫受体信号通路的进化，我们搜索了免疫受体信号接头蛋白Grb2的文昌鱼B.belcheri（Bb）同源物的相关受体。来自Belcheri肠裂解物的BbGrb2免疫沉淀物的质谱分析揭示了含有叶酸受体（FR）结构域和富含亮氨酸重复序列（LRR）的蛋白质（FrLRR）。序列和结构分析表明，FrLRR是具有预测的弯曲螺线管结构的膜蛋白。N端Fr结构域包含很少的叶酸结合位点；以下LRR区域是Slit2型LRR，在C端预测了一个GPI锚定位点。RT-PCR分析显示FrLRR是BbFR样和BbSlit2-N样基因的转录介导的融合基因。基因组DNA结构分析表明，通过将Slit2-N样基因外显子改组为FR基因，可能会产生B.belcheriFrLRR基因基因座和Floridae中的相应基因座。RT-qPCR,免疫染色和免疫印迹结果表明，FrLRR主要分布在B.belcheri肠组织中。我们进一步证明FrLRR定位于细胞膜和溶酶体。功能上,FrLRR介导和促进细菌结合和吞噬作用，FrLRR抗体阻断或Grb2敲除抑制FrLRR介导的吞噬作用。有趣的是,我们发现，人Slit2-N（hSlit2-N）也介导直接细菌结合和吞噬作用，这被Slit2-N抗体阻断或Grb2敲低抑制。一起,这些结果表明FrLRR和hSlit2-N可能作为吞噬受体通过Grb2促进吞噬,这意味着含有Slit2-N型LRR的蛋白在细菌结合和消除中起作用.
