Laboratory of Molecular Biology & Immunobiotechnology, Hellenic Pasteur Institute, Athens, Greece.
Mol Cell Biochem. 2013 Jul;379(1-2):107-13. doi: 10.1007/s11010-013-1632-8. Epub 2013 Apr 6.
EMMPRIN is a widely expressed transmembrane glycoprotein that plays important roles in many physiological and pathological processes, such as tumor invasion and metastasis. It stimulates the production of matrix metalloproteinase (MMPs) by tumor-associated fibroblasts. In the present study, our aim was to (a) to investigate if the IgII loop domain of the extracellular domain (ECD) of EMMPRIN contributes to the MMP production by fibroblasts and (b) to evaluate the significance of glycosylation in this process. For this purpose, we expressed the ECD, IgI, or IgII domains of EMMPRIN, in their glycosylated and non-glycosylated forms, in the heterologous expression systems of P. pastoris and E. coli, respectively. Dermal fibroblasts were treated with purified recombinant domains and proteins from cell extracts and supernatants were analyzed by Western blot and zymography assays. Fibroblasts treated with ECD-, IgI-, and IgII-glycosylated domains of EMMPRIN significantly stimulated the gelatinolytic activity of MMP-2, compared to untreated fibroblasts, whereas no significant effect was observed after treatment with the non-glycosylated ECD, IgI, and IgII domains. Western blot analysis from cell extracts and supernatants revealed that only the glycosylated forms were able to stimulate MMP-2 production and secretion, respectively. Quantitative PCR revealed that this effect was not attributed to transcriptional alterations. This study showed that N-glycosylation was a prerequisite for efficient MMP-2 production, with the IgII loop domain contributing significantly to this process. Perturbation of the function of IgII-EMMPRIN loop could have potential therapeutic value in the inhibition of MMP-2-dependent cancer cell invasion and metastasis.
EMMPRIN 是一种广泛表达的跨膜糖蛋白，在许多生理和病理过程中发挥重要作用，如肿瘤侵袭和转移。它刺激肿瘤相关成纤维细胞产生基质金属蛋白酶（MMPs）。在本研究中，我们的目的是：（a）研究 EMMPRIN 细胞外结构域（ECD）的 IgII 环结构域是否有助于成纤维细胞产生 MMP；（b）评估糖基化在这个过程中的意义。为此，我们分别在毕赤酵母和大肠杆菌的异源表达系统中表达了 EMMPRIN 的 ECD、IgI 或 IgII 结构域，及其糖基化和非糖基化形式。用纯化的重组结构域和细胞提取物上清液中的蛋白处理真皮成纤维细胞，通过 Western blot 和明胶酶谱分析检测。与未处理的成纤维细胞相比，用 ECD、IgI 和 IgII 糖基化结构域处理的成纤维细胞显著刺激 MMP-2 的明胶酶活性，而用非糖基化的 ECD、IgI 和 IgII 结构域处理则没有显著影响。细胞提取物和上清液的 Western blot 分析显示，只有糖基化形式才能分别刺激 MMP-2 的产生和分泌。定量 PCR 显示，这种效应不是转录改变的结果。本研究表明，N-糖基化是 MMP-2 产生的必要条件，IgII 环结构域对这一过程有重要贡献。干扰 IgII-EMMPRIN 环的功能可能在抑制 MMP-2 依赖性癌细胞侵袭和转移方面具有潜在的治疗价值。
