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

项目摘要/摘要 与正常细胞相比，癌细胞的糖基化模式发生了显著变化，但功能性的 关于癌症进展的意义还不是很清楚。这种知识差距在很大程度上是 这归因于研究细胞表面多聚糖及其相关结构的困难。这个 需要新的技术来研究糖蛋白，这是理解异常糖基化如何起作用的关键。 与癌症的发生、发展和转移有关。因此，本项目的目标是开发酶制剂 允许研究细胞表面糖蛋白的方法。 大肠杆菌分泌一种糖蛋白水解酶，分泌C1酯酶抑制物(StcE)，该酶可蛋白水解性裂解 肠上皮细胞表面的粘蛋白。该酶的初步特性证实了以前的 关于其裂解粘蛋白类型糖蛋白的能力及其对糖基化的依赖性的报道。此外，StcE的 切割基序依赖于肽序列和糖基化的存在。当它的主题被发现时 在绝大多数的粘蛋白样糖蛋白中，StcE和其他细菌粘蛋白酶可以用来打开 基础研究的新途径，同时也使治疗应用成为可能。 这些信息将被用来以两种方式询问异常糖基化。首先，自然杀伤细胞表达 抑制受体，唾液酸结合的免疫球蛋白样凝集素(Siglecs)，下调NK细胞毒性 结合含唾液酸的配体。然而，尽管知道多糖的特异性，但高亲和力 糖蛋白配体仍然难以捉摸。StcE裂解肿瘤表面Siglec-7糖蛋白配体 细胞，正如初步实验所证明的那样。因此，细胞表面蛋白将用StcE处理。劈开的 多肽将用Siglec-7结合珠富集并进行质谱分析。确认 将分析糖蛋白配体调节NK活性的能力。 其次，粘蛋白是细胞表面普遍存在的糖蛋白，具有致密的O-。 在癌症中被异常截断的糖基化。然而，目前还不清楚肿瘤是如何调节 O-糖基化的结构和位点特异性。使用像StcE这样的细菌粘液酶，这些问题可能会 克服困难。四种所谓的细菌粘蛋白酶将被表达和鉴定。MUC1和MUC16将是 从过度表达的细胞系中分离出来的细菌粘蛋白酶将被用来消化某个部位的蛋白质--和 糖链特异性的方式，多肽将用质谱学分析。此外，粘蛋白的作用 将对免疫功能的退化进行调查。 最终，如果这项提议的目标得以实现，所开发的方法将证明对实地来说是无价的。 对糖生物学的研究。此外，收集的生物信息将阐明糖基化如何起作用。 肿瘤进展，这将有助于癌症免疫治疗的设计。

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.