Development of a suite of high-specificity fucose binding reagents

项目总结 多糖具有几种独特的性质，使它们成为疾病生物标记物的极佳靶标。首先，他们的 细胞表面的定位多糖使它们成为细胞相互作用的第一个接触点，因此它们 在控制正常新陈代谢过程中至关重要。其次，细胞表面分子也是 战略性地暴露于免疫系统的监视之下，从而允许免疫识别的可能性 异常细胞。第三，在正常状态下不存在或数量较少的特定糖链结构 在疾病状态下增殖，例如癌症。最后，糖基化的变化涉及到许多蛋白质， 包括那些高度丰富的生物。因此，细胞糖基化机制的一个简单变化就可以 影响许多不同的糖结合物。有效利用和发现广泛的糖链疾病标记物 迫切需要一系列高度特异的试剂。单糖岩藻糖已在 许多疾病标记物，包括胰腺癌和前列腺癌，但很难具体检测到 现有的试剂。 利用结构引导的定向进化，我们将α-L-岩藻糖苷酶转化为高亲和力 用于检测含岩藻糖抗原的试剂。这种经过工程改造的凝集素样试剂来自于 酶被称为“Lectenz®”，与凝集素和抗体相比有几个优势。的优势 Lectenz®包括对特异性的精确定义、可调节的结合特性和重组的简便性 表达，使其在亲和纯化、Western blotting、原位组织学染色以及 活体成像。我们将产生岩藻糖结合的Lectenz®，它可以区分不同的抗原 岩藻糖结构，如刘易斯抗原与核心岩藻糖基化在哺乳动物中发现的糖基化。 糖基化检测对于充分表征和利用多糖作为特定疾病的标志是必不可少的。 然而，目前的试剂只区分Lewis X或Lewis A抗原的能力很差， 或其他刘易斯抗原或核心岩藻糖。 与抗体相比，经过改造的Lectenz®的主要优势在于，Lectenz®是针对 碳水化合物序列，但与抗体不同，它将在很大范围内识别该序列 多聚糖。此外，与基于植物凝集素的碳水化合物试剂相比，工程化的Lectenz®是衍生的 从具有精致底物特异性和低毒性的酶中提取。

PROJECT SUMMARY Glycans have several distinct properties that make them excellent targets for disease biomarkers. Firstly, their location glycans on cell surfaces makes them the first point of contact for cellular interactions, and thus they are crucial in the control of normal metabolic processes. Secondly, cell surface molecules are also strategically exposed for surveillance by the immune system allowing for the potential of immune recognition of abnormal cells. Thirdly, specific glycan structures that are not present, or are in low amounts, in normal states proliferate in disease states, such as cancer. And lastly, changes in glycosylation involve many proteins, including those that are highly abundant. Therefore, a single change in a cell’s glycosylation machinery can affect many different glycoconjugates. To effectively employ and discover glycan disease markers a wide range of highly-specific reagents are urgently needed. The monosaccharide fucose has been identified in many disease markers including pancreatic and prostate cancer, but is difficult to detect specifically with existing reagents. Using structurally-guided directed evolution, we will convert an α-L-fucosidase enzyme into high affinity reagents for the detection of fucose-containing antigens. Such engineered lectin-like reagents derived from enzymes are called “Lectenz®”, and have several advantages over lectins and antibodies. The advantages of Lectenz® include precise definition of specificity, tunable binding properties, and ease of recombinant expression, enabling their potential use in affinity purification, western blotting, in situ histological staining, and in vivo imaging. We will generate fucose-binding Lectenz® that can differentiate between various antigenic fucose structures, such as the Lewis antigens versus core fucosylation in found in mammalian glycosylation. Glycosylation detection is essential in fully characterizing and exploiting glycans as markers of specific disease states, and yet current reagents have poor abilities to discriminate between only Lewis X or Lewis A antigens, or other Lewis antigens or core fucose. The principle advantages of an engineered Lectenz® over an antibody are that the Lectenz® is specific to the carbohydrate sequence, but, in contrast to antibodies, will recognize that sequence in a broad range of glycans. Further, in contrast to carbohydrate reagents based on plant lectins, engineered Lectenz® are derived from enzymes that have exquisite substrate specificities and low toxicities.