High-specificity affinity reagents for the detection of glycan sialylation

描述(申请人提供)：葡聚糖具有几种独特的性质，使其作为疾病生物标记物的开发具有吸引力。首先，它们在细胞表面的位置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的代谢过程中起着至关重要的作用，反过来，它们也作为病原体黏附受体发挥作用。其次，特定的糖链结构不存在，或在正常状态下数量较少，在疾病状态下会增殖或改变其序列。最后，在许多蛋白质中可能会发现糖基化的变化，包括那些高度丰富的蛋白质。因此，糖链结构的正常水平的变化，如末端唾液酸，可能是疾病状态的标志。为了克服目前在发现和开发与疾病相关的多糖方面的限制，需要新的高度特异的试剂。使用结构指导的 通过基因操作，我们将肺炎链球菌的纳米B唾液酸酶转化为高特异性亲和试剂，用于检测糖肽和糖蛋白的唾液酸修饰。因为这种蛋白质具有类似凝集素的性质，但它是从一种酶中提取的，所以它被称为“Lectenz(R)”。NanB Lectenz(R)解决了疾病糖标记物检测中的一个关键需求：即一种强大且易于生产的专用于�3连接的唾液酸的试剂。该试剂可用于样品浓缩的亲和矩阵中，与现有的基于MS的方法相结合可提供连锁信息。它还可以用于组织学研究或蛋白质印迹等。与植物凝集素相比，Lectenz(R)具有许多优势：它们经过精心设计，具有高亲和力，同时保留了精致的底物专一性，可以有效地产生内源性酶，并且对于人类同源物来说，具有低毒用于体内的潜力。虽然Lectenz(R)发展的某些方面与抗体进化类似，但Lectenz(R)使用具有所需特异性的蛋白质朴素模板具有巨大的好处。

DESCRIPTION (provided by applicant): Glycans have several distinct properties that make their development as disease biomarkers appealing. Firstly, their location 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, and conversely, they function as pathogen adhesion receptors. Secondly, specific glycan structures that are not present, or are in low amounts in normal state, proliferate or alter their sequence in disease states. And, lastly, changes in glycosylation may be found in many proteins, including those that are highly abundant. Thus changes in the normal levels of glycan structures, such as terminal sialic acid, may be markers of disease states. New highly-specific reagents are required in order to overcome current limitations in the discovery and exploitation of disease-related glycans. Using structurally-guided genetic manipulations, we will convert the NanB sialidase from S. pneumococcus into a high-specificity affinity reagent for the detection of sialic acid modifications of glycopeptides and glycoproteins. Because such a protein has lectin-like properties, but is derived from an enzyme, it is called a "Lectenz(r)". A NanB Lectenz(r) addresses a key need in disease glycomarker detection: namely, a robust and easy to produce reagent specific for �3-linked sialic acid. This reagent could be employed in an affinity matrix for sample enrichment, which in conjunction with existing MS based methods could provide linkage information. It could also be employed in histological studies, or Western blots, etc. Lectenz(r) offer numerous advantages over plant lectins: they are engineered to be high affinity and yet retain the exquisite substrate specificityof the endogenous enzyme, they may be efficiently produced, and for human homologues have the potential to be employed in vivo with low toxicity. Whereas some aspects of Lectenz(r) development parallel those of antibody evolution, Lectenz(r) have the tremendous benefit of employing a protein naive template that has the desired specificity.