在节肢动物中,基因组中只有一个唐氏综合症细胞粘附分子(Dscam)的拷贝,但它可以作为许多剪接变体存在。胞外结构域中有三个高变外显子,跨膜结构域中有一个高变外显子。在中华绒螯蟹(中华绒螯蟹)中,外显子4、6和14可以产生25、34和18个选择性剪接变体,分别。在这项研究中,通过Illumina测序,我们确定了外显子6和14的其他剪接变体,因此可能存在>50,000个Dscam蛋白变体。外显子4、6和14的测序表明,细菌刺激后可变剪接发生了变化。因此,我们表达并纯化了Dscam的细胞外可变区(EsDscam-Ig1-Ig7)。外显子4.3,6.46和14.18,重组蛋白的三个可变外显子,是随机选择的。随后探讨了EsDscam-Ig1-Ig7在中华大肠杆菌免疫防御中的功能。发现EsDscam-Ig1-Ig7与革兰氏阳性金黄色葡萄球菌和革兰氏阴性副溶血性弧菌结合,但它没有表现出抗菌活性。通过促进血细胞吞噬和细菌去除,EsDscam-Ig1-Ig7还可以保护宿主免受细菌感染。这些发现突出了Dscam可变剪接的免疫活性,并揭示了比以前在中华大肠杆菌中预测的更多Dscam同工型的潜力。
In arthropods, there is only a single copy of Down Syndrome Cell Adhesion Molecule (
Dscam) in the genome, but it can exist as numerous splice variants. There are three hypervariable exons in the extracellular domain and one hypervariable exon in the transmembrane domain. In Chinese mitten crab (Eriocheir sinensis), exons 4, 6 and 14 can produce 25, 34 and 18 alternative splice variants, respectively. In this study, through Illumina sequencing, we identified additional splice variants for exons 6 and 14, hence there may be > 50,000
Dscam protein variants. Sequencing of exons 4, 6 and 14 showed that alternative splicing was altered after bacterial stimulation. Therefore, we expressed and purified the extracellular variable region of
Dscam (EsDscam-Ig1-Ig7). Exons 4.3, 6.46 and 14.18, three variable exons of the recombinant protein, were randomly selected. The functions of EsDscam-Ig1-Ig7 in immune defences of E. sinensis were subsequently explored. EsDscam-Ig1-Ig7 was discovered to bind to both Gram-positive Staphylococcus aureus and Gram-negative Vibrio parahaemolyticus, but it did not exhibit antibacterial activity. By promoting hemocyte phagocytosis and bacterial removal, EsDscam-Ig1-Ig7 can also shield the host from bacterial infection. The findings highlight the immunological activities of
Dscam alternative splicing and reveal the potential for many more
Dscam isoforms than were previously predicted in E. sinensis.