Glycophage arrays for the discovery of biomarkers in disease

描述(申请人提供)：生物体整个生命周期中的许多生物过程是由离散的糖结构及其与蛋白质和脂肪分子的相互作用所支配的。这些碳水化合物在细胞-细胞识别、黏附、细胞激活和宿主-病原体相互作用等过程中发挥着重要作用。在人类中，复杂碳水化合物合成的变化已知会导致包括癌症、逆转录病毒感染和心、肺和血液疾病在内的病理。从使用传统分析工具的研究中已经获得了大量关于碳水化合物功能的信息，但仍然需要高通量方法来表征和设计蛋白质-碳水化合物相互作用。由于各种原因，包括缺乏模板驱动的合成和糖链结构的复杂性，一直缺乏高通量的方法来表征碳水化合物/抗原相互作用。然而，即使是目前用于检测多糖结构变化的工具包，在发现癌症和艾滋病毒等疾病的新生物标记物方面也显示出巨大的潜力。最近，人们发现空肠弯曲菌天冬酰胺连接(N-连接)的蛋白糖基化系统可以功能性地转移到大肠杆菌中，从而赋予蛋白质糖基化的能力。尽管细菌N-葡聚糖在结构上不同于真核生物，但这一成就为工程糖基化反应打开了大门，这种糖基化反应模仿人类和其他高等哺乳动物中N-葡聚糖的加工过程。甘草寻求将这种“细菌糖工程”应用于诊断和治疗药物的开发。为了实现这一目标，这一特定应用的目标是合成噬菌体展示的多糖的功能微阵列(目标1)，并使用该微阵列来检测基本的蛋白质-碳水化合物相互作用(目标2)。这些研究将为今后扩大噬菌体展示多糖的多样性和分析复杂血清中的碳水化合物特征奠定必要的基础。这些研究具有重要意义，因为它们将(I)提供一种分析工具，克服目前多糖微阵列合成的瓶颈，(Ii)使未来能够合成用于研究、工业和治疗应用的新型糖结合物。公共卫生相关性：已知复合糖组装异常与多种疾病有关，包括癌症、逆转录病毒感染以及心、肺和血液疾病。基于碳水化合物的阵列系统已经成为分析这些疾病背后的碳水化合物-抗原相互作用的首选方法，然而这些阵列的未来用途关键取决于产生和回收用于固定化的各种糖聚糖的能力。这些研究的重点是开发由噬菌体展示的多聚糖组成的功能微阵列，用于探测碳水化合物与抗原的相互作用。

DESCRIPTION (provided by applicant): Many biological processes throughout the lifespan of a living organism are governed by discrete sugar structures and their interaction with protein and lipid molecules. Such carbohydrates play essential roles in processes such as cell-cell recognition, adhesion, cell activation and host- pathogen interactions. In humans, variations in the synthesis of complex carbohydrates are known to cause pathologies including cancer, retrovirus infection and disorders of the heart, lung and blood. Substantial information has been learned about carbohydrate function from studies employing traditional analytical tools, yet there remains a demand for high-throughput methods that allow for the characterization and engineering of protein-carbohydrate interactions. High-throughput methods for characterizing carbohydrate/antigen interactions have been lacking for a variety of reasons including the absence of template driven synthesis and the significant structural complexity of glycans. However, even the current toolkit for detecting changes in glycan structure has shown great potential in the discovery of novel biomarkers for diseases such as cancer and HIV. Recently, it was discovered that the Campylobacter jejuni asparagine-linked (N-linked) protein glycosylation system can be functionally transferred into E. coli, conferring the ability to glycosylate proteins. Although the bacterial N-glycan is structurally different from its eukaryotic counterparts, such an accomplishment opens the door for engineered glycosylation reactions that mimic the processing of N-glycans in humans and other higher mammals. Glycobia seeks to apply such "bacterial glycoengineering" for the development of diagnostic and therapeutic agents. Towards this goal, the objective of this particular application is to synthesize a functional microarray of phage-displayed glycans (Aim 1) and use the microarray to detect basic protein-carbohydrate interactions (Aim 2). These studies should lay the necessary foundation for future work in expanding the diversity of phage-displayed glycans and analyzing carbohydrate signatures in complex serum. These studies are significant because they should (i) provide an analytical tool that overcomes the current bottlenecks of glycan microarray synthesis and (ii) enable the future synthesis of novel glycoconjugates for research, industrial and therapeutic applications. Public Health Relevance: Abnormalities in the assembly of complex sugars are known to be associated with a multitude of diseases including cancer, retrovirus infection and disorders of the heart, lung and blood. Carbohydrate-based array systems have emerged as the preferred methodology for analyzing carbohydrate-antigen interactions underlying these disorders, however the future utility of these arrays hinges critically on the ability to generate and recover diverse glycans for immobilization. The focus of these studies is the development of functional microarrays comprised of phage- displayed glycans for probing carbohydrate-antigen interactions.