High-specificity affinity reagents for N-glycosylation site mapping and glycomics

聚糖具有几种不同的特性，使其成为疾病生物标志物的优秀靶标。首先，聚糖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，因此对正常代谢过程的控制至关重要。细胞表面分子也有策略地暴露在免疫系统的监视下，从而允许对异常细胞的免疫识别。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。最后，糖基化的变化涉及许多蛋白质，包括那些非常丰富的蛋白质。因此，细胞糖基化机制的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糖类疾病标志物，迫切需要新的糖类特异性试剂。利用计算方法指导分子进化，碳水化合物加工酶将转化为高亲和力受体蛋白，保留酶的天然特异性，但不再具有酶活性。因为这种蛋白质具有类似凝集素的特性，但它是从一种酶中提取出来的，所以我们称它们为“凝集素(r)”。与凝集素和抗体相比，Lectenz(r)作为糖组学试剂具有几个潜在的优势，包括特异性的精确定义，易于以单价形式制备，以及（对于人类同源物）最小的体内毒性。在这个提议中，肽n -聚糖酶F （PNGase F）糖加工酶将转化为含有天冬酰胺连接碳水化合物链的肽和蛋白质的高特异性亲和试剂。基于PNGase F的Lectenz(r)可以通过样品富集直接用于糖组学/蛋白质组学分析，从而促进糖基化位点定位。糖基化位点定位目前是非常繁琐的，但对于充分表征和利用聚糖作为特定疾病状态的标志物是必不可少的。工程Lectenz(r)的主要优点是Lectenz(r)对特定的碳水化合物序列具有特异性，与抗体相比，它可以在广泛的聚糖环境中识别该序列。此外，与植物凝集素相比，工程凝集素(r)来源于具有精致底物特异性和低毒性的酶。最后，由于已知有大量的碳水化合物处理酶，因此可以使用Lectenz(r)技术（正在申请专利）组装专门用于表征、监测或检测特定聚糖的亲和试剂面板。

DESCRIPTION: Glycans have several distinct properties that make them excellent targets for disease biomarkers. Firstly, the location of the glycans on the cell surface makes them the first point of contact of cellular interactions and thus crucial in the control of normal metabolic processes. Cell surface molecules are also strategically exposed for surveillance by the immune system allowing for the potential of immune recognition of abnormal cells. Secondly, specific glycan structures that are not present, or are in low amounts, in normal states proliferate in disease states. 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 new glycan-specific reagents are urgently needed. Using computational methods to guide molecular evolution, carbohydrate-processing enzymes will be converted into high affinity receptor proteins that retain the native specificity of the enzyme, but which no longer have enzyme activity. Because such a protein has lectin-like properties, but is derived from an enzyme, we are calling them " Lectenz(r)". Lectenz(r) have several potential advantages over lectins and antibodies as glycomics reagents, including precise definition of specificity, ease of preparation in a monovalent form, and (for human homologues) minimal in vivo toxicity. In this proposal, the peptide N-glycanase F (PNGase F) carbohydrate-processing enzyme will be converted into a high-specificity affinity reagent for peptides and proteins that contain asparagine-linked carbohydrate chains. The Lectenz(r) based on PNGase F may be employed directly to address the needs of glycomics/proteomics analysis through sample enrichment, thus facilitating glycosylation site-mapping. Glycosylation site mapping is currently extremely tedious to perform and yet is essential in fully characterizing and exploiting glycans as markers of specific disease states. The principle advantages of an engineered Lectenz(r) are that the Lectenz(r) is specific to a defined carbohydrate sequence, and, in contrast to antibodies, will recognize that sequence in a broad range of glycan contexts. Further, in contrast to plant lectins, engineered Lectenz(r) are derived from enzymes that have exquisite substrate specificities and low toxicities. Lastly, as there is an abundance of carbohydrate- processing enzymes known, it is possible to employ the Lectenz(r) technology (patent pending) to assemble panels of affinity reagents tailor-made for characterizing, monitoring or detecting specific glycans.