Vernet Erik, Konrad Anna, Lundberg Emma, Nygren Per-Ake, Gräslund Torbjörn
Department of Molecular Biotechnology, Royal Institute of Technology, Albanova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden.
J Immunol Methods. 2008 Sep 30;338(1-2):1-6. doi: 10.1016/j.jim.2008.06.005. Epub 2008 Jul 29.
Interference with the export of cell surface receptors can be performed through co-expression of specific affinity molecules designed for entrapment in the endoplasmic reticulum during the export process. We describe the investigation of a small (6 kDa) non-immunoglobulin-based HER2 receptor binding affibody molecule (Z(HER2:00477)), for use in affinity mediated entrapment of the HER2 receptor in the ER. Constructs encoding Z(HER2:00477) or a control affibody protein, with or without ER-retention peptide extensions (KDEL), were expressed in the HER2 over-expressing cell line SKOV-3. Intracellular expression of the full-length affibody constructs could be confirmed by probing cell extracts by Western blotting. Confocal immunofluorescence microscopy experiments showed extensive co-localization of the HER2 receptor and Z(HER2:00477)-KDEL in the ER, whereas the use of a KDEL-extended control affibody molecule resulted in distinct and separate signals from cell surface-localized HER2 receptor and ER-localized affibody protein. This indicated a capability of the Z(HER2:00477)-KDEL fusion protein to functionally interfere with the export process of HER2 receptor in a specific manner. Using flow cytometry and cell proliferation analyses, it could be shown that expression of the Z(HER2:00477)-KDEL fusion construct in the SKOV-3 cell line resulted both in a marked reduction in cell surface level of HER2 receptors and that the cell population doubling time was significantly increased. Expression of the Z(HER2:00477)-KDEL fusion protein in additional cell lines of different origin and with different expression levels of endogenous HER2 receptor compared to SKOV-3, also resulted in depletion of the cell surface levels of HER2 receptor. This indicated upon a general ability of the Z(HER2:00477)-KDEL fusion protein to functionally interfere with the export process of HER2.
通过共表达特定的亲和分子来干扰细胞表面受体的输出，这些亲和分子设计用于在输出过程中被困在内质网中。我们描述了一种基于非免疫球蛋白的小（6 kDa）HER2受体结合亲和体分子（Z(HER2:00477)）的研究，用于在ER中亲和介导HER2受体的滞留。编码Z(HER2:00477)或对照亲和体蛋白的构建体，带有或不带有内质网滞留肽延伸（KDEL），在HER2过表达细胞系SKOV-3中表达。通过蛋白质印迹法检测细胞提取物，可以确认全长亲和体构建体的细胞内表达。共聚焦免疫荧光显微镜实验表明，HER2受体与ER中的Z(HER2:00477)-KDEL广泛共定位，而使用KDEL延伸的对照亲和体分子则导致细胞表面定位的HER2受体和ER定位的亲和体蛋白产生明显且分离的信号。这表明Z(HER2:00477)-KDEL融合蛋白能够以特定方式在功能上干扰HER2受体的输出过程。使用流式细胞术和细胞增殖分析，可以表明Z(HER2:00477)-KDEL融合构建体在SKOV-3细胞系中的表达导致HER2受体的细胞表面水平显著降低，并且细胞群体倍增时间显著增加。与SKOV-3相比，在不同来源且内源性HER2受体表达水平不同的其他细胞系中表达Z(HER2:00477)-KDEL融合蛋白，也导致HER2受体的细胞表面水平降低。这表明Z(HER2:00477)-KDEL融合蛋白具有在功能上干扰HER2输出过程的一般能力。
