家蚕EGFR配体的鉴定和验证

Identification and characterization of epidermal growth factor receptor ligands in Bombyx mori

摘要:

表皮生长因子受体 (EGFR) 是广泛存在于后生动物中的多功能受体，其配体的种类、活化方式、配体与EGFR之间的相互作用以及激活的信号通路在哺乳动物中研究得较为深入。而非脊椎动物中，EGFR配体在各物种间差异较大，目前缺乏对除果蝇以外的其他昆虫EGFR配体的认识。通过同源比对、结构域预测、mRNA翻译起始序列分析和系统进化树构建，在家蚕中鉴定到2个EGFR的配体，命名为BmEGF-1和BmEGF-2。BmEGF-1与果蝇Spitz有较高的同源性和一致的Rhomboid识别序列，BmEGF-2为Vein的同源分子。经原核表达和纯化获得了BmEGF-1胞外区段，利用Sf9细胞分泌BmEGFR胞外区段，并通过pull-down实验验证了两者之间存在相互作用。在BmE细胞中表达BmEGF-1后，通过Western blotting检测到ERK和p38 MAPK的磷酸化水平增强，说明其不仅能激活经典的ERK信号通路，还可能通过p38 MAPK信号通路参与其他生理过程，为进一步研究EGFR配体在家蚕中的生物学功能提供了参考。

Abstract:

Epidermal growth factor receptor (EGFR) is a multi-functional receptor distributed throughout the metazoa. Study on its ligands so far remained mainly on mammals, including how ligands are processed into active forms, their interaction with EGFR, and the signaling pathway they induce. However, in invertebrates, ligands are more divergent among species. Currently, except for Drosophila, less is known about the insect EGFR ligands. Here, we identified two EGFR ligands in Bombyx mori by homology search, domain prediction, analysis of the potential translation initiation sequence and construction of phylogenetic tree, termed as BmEGF-1 and BmEGF-2. BmEGF-1 shows the greatest similarity to Drosophila Spitz and their Rhomboid-recognition motifs are highly identical. BmEGF-2 is a homolog to Drosophila Vein. Then we purified BmEGF-1 extracellular domain expressed in E. coli, and performed pull-down assay with BmEGFR extracellular domain secreted by Sf9 cells. The result confirmed their interaction. Lastly, we found the phosphorylation level of ERK and p38 MAPK was elevated after expression of BmEGF-1 in BmE cells, which suggested that BmEGF-1 is not only able to activate the canonical ERK signaling pathway, but may participate in other cellular processes by inducing p38 MAPK signaling pathway. Our study provides reference to further study of the biological function of BmEGF in silkworm.