Impact of Tumor-Associated Glycosylation on the Multi-Functionality of TIMP-1(Tissue Inhibitor of Metalloproteinases-1)

为了确定癌症治疗的新靶点，有必要了解单个蛋白质在肿瘤进展和转移的特定过程中的确切作用。在基因组学时代之外，现在越来越多的人意识到，由于翻译后修饰，由一个基因表达产生的蛋白质变体确实表现出多种不同的功能(多向性)。蛋白质N-糖基化是真核细胞中以物种特异性方式发生的最通用的翻译后修饰，对调节大多数与肿瘤相关的细胞过程至关重要，如信号转导、免疫调节或细胞-基质相互作用。最近，与肿瘤相关的蛋白质“糖链”的变化被认为是确定癌症所有特征的关键“使能特征”。在小鼠的实验装置中或在体外，当未确定糖基化模式的重组蛋白用于功能分析时，很少考虑糖糖的物种和肿瘤细胞特异性。金属蛋白酶组织抑制因子-1(TIMP-1)是一种在多种肿瘤相关的病理生理过程中发挥重要作用的分泌型多功能因子，是一种在生理条件下表现出特异性N-糖基化模式的糖蛋白。来自癌症患者的糖基化修饰的TIMP-1显示出降低的抗蛋白分解活性，从而介导了促肿瘤作用。在初步实验中，我们还可以证明人TIMP-1的非典型性TIMP-1的信号功能也受到糖基化的影响。在目前的项目中，我们的目标是揭示糖基化对人TIMP-1多功能的影响。我们将评估结合特征、信号活性和抗蛋白分解功能的宏观-异质性糖基化依赖关系。此外，我们的目标是在胰腺癌的背景下，通过N-糖分析来确定迄今未知的人类TIMP-1的疾病相关糖基化模式。在未来，这些发现可以被用于诊断和治疗方法，并将有助于理解TIMP-1在癌症进展中相反的功能。这项关于TIMP-1的研究的更广泛的意义和概念性进展是，糖基化对(多)功能的影响可能被翻译成其他临床相关的糖蛋白，包括细胞因子和白细胞介素2。

In order to define new targets for cancer therapy, it is necessary to understand the exact role of individual proteins in specific processes of tumor progression and metastasis. Beyond the era of genomics, it is now increasingly appreciated that protein variants resulting from expression from one gene do exhibit a multitude of differential functions (pleiotropism) as a consequence of post-translational modifications. Protein N-glycosylation is the most versatile post-translational modification occurring in a species-specific manner in eukaryotic cells and crucially contributes to the regulation of most diverse tumor-relevant cellular processes, such as signal transduction, immuno-modulation or cell-matrix interactions. Tumor-associated change of the ‘glycome’ of proteins was recently appreciated as crucial ‘Enabling Characteristic’ for the establishment of all hallmarks of cancer. Species- and tumor cell-specificity of the glycome is rather rarely considered in experimental set-ups in mice or in vitro, when recombinant proteins with undefined glycosylation pattern are employed in functional assays. Tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted multi-functional factor playing important roles in numerous cancer-associated patho-physiological processes, is a glycoprotein displaying specific N-glycosylation patterns under physiological conditions. Glycosylation-modified TIMP-1 from cancer patients exhibits reduced anti-proteolytic activity, thereby mediating pro-tumorigenic effects. In preliminary experiments, we could show that also the non-canonical tetraspanin (CD63)-mediated signaling function of human TIMP-1 is influenced by glycosylation. In the current project, we aim to unravel the impact of glycosylation on the multi-functionality of human TIMP-1. We will evaluate the macro-heterogeneous glycosylation-dependency of binding features, signaling activity, and anti-proteolytic function. Moreover, we aim to identify so-far unknown disease-associated glycosylation patterns of human TIMP-1 by N-glycome analysis in the context of pancreatic cancer. In the future these findings can be exploited for diagnostic and therapeutic approaches and will help to understand the contrary functions of TIMP-1 in cancer progression. The broader significance and conceptual advance of this study on TIMP-1 is that the impact of glycosylation on (multi-) functionality may be translated to other clinically relevant glycoproteins including cytokines and interleukins.