Development of a suite of high-specificity fucose binding reagents

一套高特异性岩藻糖结合试剂的开发

• 批准号: 9909159
• 负责人: Loretta Yang
• 金额: $ 22.5万
• 依托单位: GLYCOSENSORS AND DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9909159
• 关键词: Abnormal Cell; Active Sites; Address; Affect; Affinity; Affinity Chromatography; Amino Acids; Antibodies; Antigens; Autoimmune Diseases; Binding; Biological Markers; Carbohydrate Sequence; Carbohydrates; Cell surface; Cells; Chemicals; Complex; Computer Analysis; Data; Detection; Development; Directed Molecular Evolution; Disease; Disease Marker; Engineering; Enzymes; Epitopes; Free Energy; Fucose; Fucosidase; Glycoconjugates; Glycoproteins; Glycoside Hydrolases; Human; Immune; Immune system; Immunologic Surveillance; In Situ; Individual; Interferometry; Lectin; Libraries; Ligands; Link; Location; Malignant Neoplasms; Malignant neoplasm of pancreas; Malignant neoplasm of prostate; Mass Spectrum Analysis; Measurement; Metabolism; Methods; Monosaccharides; Mutagenesis; Pancreas; Phase; Plant Lectins; Polymers; Polysaccharides; Positioning Attribute; Preparation; Proliferating; Property; Prostate; Protein Subunits; Proteins; Reagent; Recombinants; Rheumatoid Arthritis; Sampling; Site; Site-Directed Mutagenesis; Specificity; Spectrometry; Structure; Substrate Specificity; Surface; Techniques; Thermotoga maritima; Tissue Sample; Toxic effect; Western Blotting; Yeasts; arm; base; cancer type; carbohydrate binding protein; design; differential expression; genetic linkage analysis; genetic manipulation; glycosylation; glycosyltransferase; histological stains; improved; in vivo; in vivo imaging; insight; molecular dynamics; mutant; novel; preference; response; scaffold; screening

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.

项目摘要 Glycans具有几种不同的特性，使其成为疾病生物标志物的绝佳目标。首先，他们 细胞表面上的地理聚糖使它们成为细胞相互作用的第一个接触点，因此它们是 对于控制正常代谢过程至关重要。其次，细胞表面分子也是 免疫系统在策略上暴露于监视，允许免疫认识的潜力 异常细胞。第三，在正常状态下不存在或低量的特定聚糖结构 在疾病状态（例如癌症）中扩散。最后，糖基化的变化涉及许多蛋白质， 包括那些高度丰富的。因此，细胞糖基化机械的单一更改可以 影响许多不同的糖缀合物。有效利用和发现宽阔的聚糖疾病标记 迫切需要一系列高度特异性试剂。在 许多疾病标志物在内，包括胰腺癌和前列腺癌，但很难专门检测 现有试剂。 使用结构引导的定向进化，我们将将α-l-糖粉酶转化为高亲和力 检测含岩藻糖的抗原的试剂。这种工程讲座的试剂来自 酶称为“Lectenz®”，比讲座和抗体具有多个优点。优势 Lectenz®包括特异性的精确定义，可调节结合特性以及重组易于 表达，使其在亲和力纯化，蛋白质印迹，原位组织学染色以及 体内成像。我们将生成可以区分各种抗原的Fucose结合Lectenz® 在哺乳动物糖基化中发现的岩藻糖结构，例如刘易斯抗原与核心构成。 糖基化检测对于充分表征和利用聚糖作为特定疾病的标记至关重要 各州，但目前的试剂的能力较差，仅区分刘易斯X或刘易斯A抗原， 或其他刘易斯抗原或核心岩岩。 工程Lectenz®比抗体的主要优点是，Lectenz®是特定于 碳水化合物序列，但与抗体相反，将认识到该序列 聚糖。此外，与基于植物讲座的碳水化试剂相比，衍生了工程的Lectenz® 来自具有独家底物规范和低毒性的酶。