High-specificity affinity reagents for the detection of glycan sialylation

描述（由申请人提供）：聚糖具有几种不同的特性，使其成为具有吸引力的疾病生物标志物。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代谢过程中至关重要，相反，它们作为病原体粘附受体发挥作用。其次，在正常状态下不存在或含量低的特定聚糖结构在疾病状态下增殖或改变其序列。最后，可以在许多蛋白质中发现糖基化的变化，包括那些非常丰富的蛋白质。因此，正常水平的聚糖结构（如末端唾液酸）的变化可能是疾病状态的标志。为了克服目前在发现和利用疾病相关聚糖方面的限制，需要新的高特异性试剂。使用structurally-guided

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.