An enzymatic approach to study cancer-associated cell-surface glycoproteins: exploration of mucin-degrading bacterial metalloproteases

项目总结/摘要 与正常细胞相比，癌细胞具有显著改变的糖基化模式，但功能性糖基化模式与癌细胞的糖基化模式不同。 关于癌症进展的重要性还没有很好地理解。这种知识差距在很大程度上是 这归因于与研究细胞表面聚糖及其相关结构相关的困难。的 需要新的技术来研究糖蛋白是至关重要的，以了解异常糖基化如何有助于 癌症的发生、发展和转移。因此，本项目的目标是开发酶促 允许研究细胞表面糖蛋白的方法。 E.大肠杆菌分泌一种蜜环菌蛋白酶，即C1-酯酶抑制剂（StcE）的分泌蛋白酶， 肠上皮细胞表面的粘蛋白。这种酶的初步表征证实了以前的 报道了其切割粘蛋白型糖蛋白的能力及其对糖基化的依赖性。此外，StcE 切割基序依赖于肽序列和糖基化的存在。因为它的主题被发现 在绝大多数粘蛋白样糖蛋白中，StcE和其他细菌粘蛋白酶可用于打开 基础研究的新途径，同时也使治疗应用。 该信息将用于以两种方式询问异常糖基化。首先，自然杀伤细胞表达 抑制性受体，唾液酸结合免疫球蛋白样凝集素（Siglecs），下调NK细胞毒性 在结合它们的含唾液酸的配体时。然而，尽管了解聚糖特异性，高亲和力的 糖蛋白配体仍然难以捉摸。StcE切割肿瘤表面的Siglec-7糖蛋白配体 细胞，如初步实验所示。因此，细胞表面蛋白将用StcE处理。裂解 肽将用Siglec-7缀合的珠富集并通过质谱分析。证实 分析糖蛋白配体调节NK活性的能力。 其次，粘蛋白是细胞表面上普遍存在的糖蛋白，其特征在于具有致密的O- 在癌症中异常截短的糖基化。然而，目前尚不清楚肿瘤如何调节 O-糖基化的结构和位点特异性。使用像StcE这样的细菌粘蛋白酶，这些问题可以得到解决。 克服四个假定的细菌粘蛋白酶将被表达和表征。MUC 1和MUC 16将是 从过表达细胞系中分离的细菌粘蛋白酶将用于消化位点中的蛋白质， 聚糖特异性方式，肽将用质谱法分析。此外，粘蛋白的作用 将研究对免疫功能的降解。 最后，如果本建议的目标得以实现，所开发的方法将证明对该领域是非常宝贵的 糖生物学此外，收集的生物学信息将阐明糖基化如何有助于 这将有助于癌症免疫疗法的设计。

Project Summary/Abstract Cancer cells have significantly altered glycosylation patterns compared to normal cells, but the functional significance with regard to cancer progression is not well understood. This gap in knowledge is largely attributable to the difficulties associated with studying cell-surface glycans and their associated structures. The need for new techniques to study glycoproteins is crucial to understand how aberrant glycosylation contributes to the onset, progression, and metastasis of cancer. Thus, the goal of this project is to develop enzymatic methods that allow for the study of cell-surface glycoproteins. E. coli secretes a mellaloprotease, secreted protease of C1-esterase inhibitor (StcE) that proteolytically cleaves mucin proteins on the surface of gut epithelial cells. Initial characterization of this enzyme confirmed previous reports of its ability to cleave mucin-type glycoproteins and its dependency on glycosylation. Further, StcE’s cleavage motif is dependent on both peptide sequence and the presence of glycosylation. As its motif is found within the vast majority of mucin-like glycoproteins, StcE and other bacterial mucinases can be used to open new avenues of basic research while also enabling therapeutic applications. This information will be used to interrogate aberrant glycosylation in two ways. First, natural killer cells express inhibitory receptors, sialic acid-binding immunoglobulin-like lectins (Siglecs), that downregulate NK cytotoxicity upon binding their sialic acid containing ligands. However, despite knowledge of glycan specificity, high-affinity glycoprotein ligands have remained elusive. StcE cleaves Siglec-7 glycoprotein ligands on the surface of tumor cells, as demonstrated by preliminary experiments. Thus, cell surface proteins will be treated with StcE. Cleaved peptides will be enriched with Siglec-7 conjugated beads and analyzed by mass spectrometry. Confirmed glycoprotein ligands will be analyzed for their ability to modulate NK activity. Secondly, mucin proteins are prevalent glycoproteins on the cell surface and are characterized by dense O- glycosylation that is abnormally truncated in cancer. However, it is not understood how tumors modulate structures and site-specificity of O-glycosylation. Using bacterial mucinases like StcE, these issues can be overcome. Four purported bacterial mucinases will be expressed and characterized. MUC1 and MUC16 will be isolated from overexpressing cell lines, bacterial mucinases will be used to digest the proteins in a site- and glycan-specific manner, and peptides will be analyzed with mass spectrometry. Additionally, the role of mucin degradation on immune function will be investigated. Ultimately, should the goals of this proposal be attained, the methods developed will prove invaluable to the field of glycobiology. Additionally, the biological information gathered will shed light on how glycosylation contributes to tumor progression, which will help in cancer immunotherapy design.