Funahashi Shin-Ichi, Suzuki Yasunori, Nakano Kiyotaka, Kawai Shigeto, Suzuki Masami
Forerunner Pharma Research Co., Ltd., Komaba Open Laboratory, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan.
J Biochem. 2017 Apr 1;161(4):361-368. doi: 10.1093/jb/mvw077.
Leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6) is a seven-pass transmembrane protein known to be a marker of stem cells in several organs. To deepen our understanding of the cell biology of LGR6-positive cells, including stem cells, we generated monoclonal antibodies (mAbs) against human LGR6. DNA immunization followed by whole-cell immunization with LGR6-expressing transfectants was performed to obtain mAbs that recognized the native form of LGR6. Hybridomas were screened by flow cytometry using LGR6-transfected cells. Because the molecules of LGR4, LGR5, and LGR6 are 50% homologous at the amino acid level, specificity of the mAbs was confirmed by transfectants expressing LGR4, LGR5, or LGR6. Three LGR6-specific mAbs were generated. Two of the three mAbs (designated 43A6 and 43D10) recognized the large N-terminal extracellular domain of LGR6, and competitively blocked the binding of R-spondin 1, which is known to be the ligand for LGR6. The other mAb, 43A25, recognized the seven-pass transmembrane domain of LGR6, and was able to be used for immunoblot analysis. In addition, mAbs 43A6 and 43D10 detected endogenous expression of LGR6 in cancer cell lines. We expect that our mAbs will contribute to widening our understanding of LGR6-positive cells in humans.
富含亮氨酸重复序列的G蛋白偶联受体6（LGR6）是一种七次跨膜蛋白，已知是多个器官中干细胞的标志物。为了加深我们对包括干细胞在内的LGR6阳性细胞的细胞生物学的理解，我们制备了针对人LGR6的单克隆抗体（mAb）。通过DNA免疫，随后用表达LGR6的转染子进行全细胞免疫，以获得识别LGR6天然形式的mAb。使用LGR6转染细胞通过流式细胞术筛选杂交瘤。由于LGR4、LGR5和LGR6的分子在氨基酸水平上有50%的同源性，因此通过表达LGR4、LGR5或LGR6的转染子确认了mAb的特异性。产生了三种LGR6特异性mAb。三种mAb中的两种（命名为43A6和43D10）识别LGR6的大的N端细胞外结构域，并竞争性阻断已知为LGR6配体的R-spondin 1的结合。另一种mAb 43A25识别LGR6的七次跨膜结构域，并可用于免疫印迹分析。此外，mAb 43A6和43D10检测到癌细胞系中LGR6的内源性表达。我们期望我们的mAb将有助于拓宽我们对人类LGR6阳性细胞的理解。
