Development of a suite of high-specificity fucose binding reagents

项目摘要 聚糖具有几种独特的性质，使其成为疾病生物标志物的理想靶标。首先，他们的 在细胞表面上的位置聚糖使它们成为细胞相互作用的第一个接触点，因此它们 在控制正常代谢过程中至关重要。其次，细胞表面分子也是 策略性地暴露于免疫系统的监视中，从而允许免疫识别的潜力， 异常细胞第三，在正常状态下不存在或少量存在的特定聚糖结构 在疾病状态下增殖，例如癌症。最后，糖基化的变化涉及许多蛋白质， 包括那些非常丰富的。因此，细胞糖基化机制中的一个单一变化可以 影响许多不同的糖缀合物。为了有效地利用和发现聚糖疾病标志物， 迫切需要一系列高度特异性的试剂。单糖岩藻糖已被确定， 许多疾病标志物，包括胰腺癌和前列腺癌，但很难特异性检测， 现有试剂 利用结构导向的定向进化，我们将α-L-岩藻糖苷酶转化为高亲和力的 用于检测含岩藻糖抗原的试剂。这种工程化的凝集素样试剂衍生自 酶被称为“Lectenz®"，并且具有优于凝集素和抗体的几个优点。的优点 Lectenz®包括特异性的精确定义，可调的结合特性，以及重组的容易性。 表达，使它们能够在亲和纯化、蛋白质印迹、原位组织学染色和免疫印迹中潜在地使用。 体内成像。我们将产生岩藻糖结合Lectenz®，它可以区分各种抗原 岩藻糖结构，如刘易斯抗原与哺乳动物糖基化中发现的核心岩藻糖基化。 糖基化检测对于充分表征和利用聚糖作为特定疾病的标志物至关重要 然而，目前的试剂仅区分刘易斯X或刘易斯A抗原的能力很差， 或其它刘易斯抗原或核心岩藻糖。 工程化的Lectenz®相对于抗体的主要优点是Lectenz®对抗体是特异性的。 碳水化合物序列，但是，与抗体相反，将在广泛范围内识别该序列。 聚糖此外，与基于植物凝集素的碳水化合物试剂相反，工程化的Lectenz® 来自于具有精确的底物特异性和低毒性的酶。

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.