Engineered probes for sialoglycan detection

项目摘要/摘要 唾液酸聚糖可能是很差的抗原，这意味着开发有效的抗体来治疗 他们的侦测。相反，唾液酸聚糖图谱在很大程度上依赖于需要专业知识的质谱学 以及为数不多的实验室可用的仪器。最近在这一领域的一项努力是收获广度 在自然产生的唾液酸聚糖结合蛋白中发现的选择性，以便开发糖检测 蛋白质。这一策略确定了许多唾液酸聚糖结合蛋白，特别是那些与高密度脂蛋白 对唾液酸化T抗原(Sta)的亲和力和狭义选择性。然而，主要差距仍然存在于 被识别的唾液酸聚糖。 在这里，我们关注的是唾液酸多聚糖，它们可能在细胞中含量丰富，或者在疾病中普遍存在，但缺乏 用于检测它们的实用探针，特别是α2，3连接和α2，6连接的唾液酸聚糖。我们建议创建 通过剪裁现有唾液聚糖结合蛋白的特异性来识别这些多糖的探针 基于结构的蛋白质工程。 在目标1中，我们设计了细菌Siglec样黏附素，以创建一个三联体和四联体的探针库。 糖类，每个糖类与一个α2，3连接的唾液酸聚糖结合，亲和力高，选择性窄。 在目标2中，我们将工程学原理应用于α2，3连接唾液酸聚糖探针的开发和 关注α2，6连接唾液酸基TN抗原二糖，这是癌症的生物标志物。 在目标3中，我们评估了这些探针在测量人血浆中多聚糖方面的效用，并进行了交叉验证 这些都是通过亲和捕获和质谱分析得到的。区分葡聚糖丰度和谱带的能力 健康捐赠者与癌症患者的对比将被包括在这一目标中。成功的探测将被分发 用于凝集素阵列和低通量分析。

PROJECT SUMMARY/ABSTRACT Sialoglycans can be poor antigens, meaning that it can be challenging to develop effective antibodies for their detection. Instead, sialoglycan mapping heavily relies upon mass spectrometry, which requires expertise and instrumentation available to few laboratories. One recent push in the field has been to harvest the breadth of selectivity found within naturally-occurring sialoglycan-binding proteins in order to develop glycan-detecting proteins. This strategy identified a number of sialoglycan-binding proteins, particularly those that bind with high affinity and narrow selectivity to sialyl-T antigen (sTa). However, major gaps remain in the spectrum of the sialoglycans that are recognized. Here, we focus on sialoglycans that are likely abundant on cells or that are prevalent in disease but that lack practical probes for their detection, specifically α2,3 linked and α2,6 linked sialoglycans. We propose to create probes that recognize these glycans by tailoring the specificity of existing sialoglycan binding proteins using structure-based protein engineering. In Aim 1, we engineer the bacterial Siglec-like adhesins to create a library of probes for tri- and tetra- saccharides, each of which binds one α2,3 linked sialoglycan with high affinity and narrow selectivity. In Aim 2, we apply engineering principles to the development of probes for α2,3 linked sialoglycans and focus on the α2,6 linked sialyl Tn antigen disaccharide, a biomarker for cancer. In Aim 3, we evaluate the utility of these probes in measuring glycans in human plasma, and cross-validated these by affinity capture and mass spectrometry. The ability to distinguish glycan abundance and repertoire in healthy donors versus cancer patients will be included as a part of this aim. Successful probes will be distributed for use both in lectin arrays and in low-throughput assays.