Division of Innate Immunity, Department of Microbiology and Immunology.
Division of Clinical Genome Research, Advanced Clinical Research Center.
Int Immunol. 2017 Aug 1;29(8):347-355. doi: 10.1093/intimm/dxx044.
The Toll family of receptors sense microbial products and activate a defense response. The molecular machinery required for the TLR response is not yet fully understood. In the present study, we used a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CAS9 screening system to study TLR responses. We employed a cell line expressing TLR with an NF-κB-driven GFP reporter. The cell line was transduced with a guide RNA (gRNA) library and stimulated with TLR ligands. The cells impaired in GFP induction were sorted, and gRNAs were sequenced. Identified genes were ranked according to the count of sequence reads and the number of gRNA target sites. The screening system worked correctly, as molecules that were already known to be required for the TLR response were identified by the screening. Furthermore, this system revealed that the oligosaccharide transferase complex (OSTC) mediating co-translational glycosylation was required for TLR5, 7 and 9 responses. Protein expression of TLR5, but not an irrelevant molecule (CD44), was abolished by the lack of OSTC, suggesting the essential role of glycosylation in TLR5 protein stability. These results demonstrate that the screening system established here is able to reveal molecular mechanisms underlying the TLR response.
Toll 受体家族感知微生物产物并激活防御反应。TLR 反应所需的分子机制尚不完全清楚。在本研究中，我们使用了一种聚类、规则间隔、短回文重复序列 (CRISPR)/CAS9 筛选系统来研究 TLR 反应。我们使用了一种表达 TLR 的细胞系，该细胞系带有 NF-κB 驱动的 GFP 报告基因。该细胞系被转导了一个 gRNA 文库，并受到 TLR 配体的刺激。GFP 诱导受损的细胞被分选，gRNA 被测序。根据序列读数的数量和 gRNA 靶位点的数量对鉴定出的基因进行排序。该筛选系统运行正常，因为筛选出的已知对 TLR 反应必需的分子。此外，该系统还表明，介导共翻译糖基化的寡糖转移酶复合物 (OSTC) 是 TLR5、7 和 9 反应所必需的。OSTC 的缺乏导致 TLR5 的蛋白表达而不是无关分子 (CD44) 的表达被废除，这表明糖基化在 TLR5 蛋白稳定性中起着至关重要的作用。这些结果表明，这里建立的筛选系统能够揭示 TLR 反应的分子机制。
