Noda Natsumi, Ozawa Takeaki
Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
J Cell Sci. 2022 Mar 15;135(6). doi: 10.1242/jcs.259314. Epub 2022 Mar 17.
Cluster of differentiation 44 (CD44) is a single-pass transmembrane glycoprotein that is a widely distributed cell-surface adhesion molecule. CD44 undergoes ectodomain cleavage by membrane-associated metalloproteinases in breast cancer cells. Cleavage plays a critical role in cancer cell migration by mediating the interaction between CD44 and the extracellular matrix. To explore inhibitors of CD44 ectodomain cleavage, we developed two bioluminescent sensors for the detection of CD44 ectodomain cleavage. The sensors were designed as two-transmembrane proteins with split-luciferase fragments, one of which was cyclized by protein trans-splicing of a DnaE intein. These two sensors emit light by the cyclization or the spontaneous complementation of the luciferase fragments. The luminescence intensities decreased upon cleavage of the ectodomain in breast cancer cells. The sensors revealed that castanospermine, an α-glucosidase inhibitor, suppressed the ectodomain cleavage of endogenous CD44 in breast cancer cells. Castanospermine also inhibited breast cancer cell invasion. Thus, the sensors are beneficial tools for evaluating the effects of different inhibitors.
分化簇44（CD44）是一种单次跨膜糖蛋白，是一种广泛分布的细胞表面粘附分子。在乳腺癌细胞中，CD44会被膜相关金属蛋白酶进行胞外域切割。这种切割通过介导CD44与细胞外基质之间的相互作用，在癌细胞迁移中发挥关键作用。为了探索CD44胞外域切割的抑制剂，我们开发了两种用于检测CD44胞外域切割的生物发光传感器。这些传感器被设计为带有分裂荧光素酶片段的双跨膜蛋白，其中一个通过DnaE内含肽的蛋白质反式剪接进行环化。这两种传感器通过荧光素酶片段的环化或自发互补来发光。在乳腺癌细胞中，胞外域切割后发光强度降低。这些传感器表明，α-葡萄糖苷酶抑制剂栗精胺可抑制乳腺癌细胞中内源性CD44的胞外域切割。栗精胺还抑制乳腺癌细胞的侵袭。因此，这些传感器是评估不同抑制剂效果的有益工具。
