Laboratory of Molecular Biotechnology, University Medical Center Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
J Biol Chem. 2010 Nov 26;285(48):37823-37. doi: 10.1074/jbc.M110.136408. Epub 2010 Sep 24.
We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.
我们通过酵母双杂交筛选研究了 ECM1（细胞外基质蛋白 1）的相互作用，鉴定出跨膜蛋白 II 型 PLSCR1（磷脂 scramblase 1）是其结合伴侣。这种相互作用随后通过体外和体内共免疫沉淀实验得到证实，并且通过 GST 标记的 ECM1a 片段进行的其他下拉实验将这种相互作用定位到 ECM1a 的串联重复区。此外，免疫组织化学染色显示，在人皮肤的基底表皮细胞层中，ECM1 和 PLSCR1 部分重叠。此外，在人皮肤等效物中，两种蛋白质均以真皮成纤维细胞依赖性方式在基膜处表达。接下来，对超薄人皮肤切片进行免疫金电子显微镜检查显示，ECM1 和 PLSCR1 共定位于细胞外基质中，并且使用针对 ECM1 或 PLSCR1 的抗体交联到磁性免疫珠上，我们能够证明在人皮肤提取物中存在 PLSCR1-ECM1 相互作用。此外，尽管 ECM1 是通过内质网/高尔基体依赖途径分泌的，但 PLSCR1 从 HaCaT 角质形成细胞中的释放是通过脂筏依赖机制发生的，并沉积在细胞外基质中。总之，我们在此证明 PLSCR1 与人皮肤真皮表皮交界处的 ECM1a 的串联重复区相互作用，并首次提供实验证据表明 PLSCR1 通过非典型分泌途径进行分泌。这些数据表明 PLSCR1 是一种多功能蛋白，既可以在细胞内发挥作用，也可以在细胞外发挥作用，并且与 ECM1 一起可能在人皮肤中发挥调节作用。
