Engineering of glycosyltransferases to obtain glycan binding proteins

糖基转移酶工程以获得聚糖结合蛋白

• 批准号: 10259786
• 负责人: SRIRAM NEELAMEGHAM
• 金额: $ 19.46万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10259786
• 关键词: Address; Adhesions; Affinity; Animal Lectins; Apoenzymes; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Biological Process; CRISPR library; CRISPR/Cas technology; Carbohydrates; Cell Adhesion; Cell Line; Cell surface; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Complement; Complex; Databases; Detection; Diabetes Mellitus; Diagnostic; Directed Molecular Evolution; Disease; Docking; Engineering; Enzyme Tests; Enzymes; Epitopes; Exhibits; Extracellular Protein; Family; Frequencies; Functional disorder; Future; Glycoside Hydrolases; Goals; Health; Human; Immune response; Incubated; Individual; Infection; Inflammation; Investigation; Kinetics; Knock-out; Lectin; Libraries; Ligand Binding; Ligands; Link; Malignant Neoplasms; Measures; Modeling; Molecular Conformation; Monosaccharides; Motion; Mutate; Mutation; Neoplasm Metastasis; Neuraminidase; Outcome; Pathologic Processes; Pathway interactions; Physiology; Plant Lectins; Polysaccharides; Post-Translational Protein Processing; Principal Component Analysis; Process; Property; Protein Engineering; Protein-Carbohydrate Interaction; Publishing; Reagent; Research; Role; ST6Gal I; Sampling; Sialic Acids; Sialyltransferases; Signal Transduction; Specificity; Structural Models; Structure; Surface; System; Technology; Testing; Tissues; Ursidae Family; Vertebral column; Yeasts; base; carbohydrate receptor; cell growth; cell type; design; enzyme structure; glycosylation; glycosyltransferase; improved; in vivo; knockout gene; lactosamine; models and simulation; molecular dynamics; molecular modeling; mutant; novel; overexpression; pathogen; screening; sialylation; success; targeted delivery; tumor

Most human extracellular proteins are post-translationally modified by N-linked glycans attached to Asn, and O- linked glycans attached to Ser/Thr. Such glycans control or fine-tune a number of biological processes including cell growth, differentiation, cell adhesion, and signaling. As a result, changes in glycosylation are also associated with mammalian pathophysiological processes like tumor metastasis, host-pathogen recognition, inflammation etc. An important impediment to understanding the role of protein-carbohydrate interactions in human health and disease is the lack of a streamlined technology to rapidly and accurately characterize glycans in arbitrary cell/ tissue systems. Carbohydrate binding lectins are commonly used to characterize cell-surface glycans, but the binding specificity and affinity of natural plant and animal lectins is poor. There has also been some success in developing novel glycan binding proteins (GBPs) by engineering, for example, sialidases in order to recognize sialic acid containing glycans, but these reagents typically only bind terminal residues with less specificity for the glycan backbone. In this proposal, we describe an alternative approach to engineering GBPs starting with glycosyltransferases (glycoT), particularly with a focus on the sialyltransferase (ST) family. We hypothesize that engineering this class of enzymes may enable specific detection of larger glycan structures with high specificity. In this regard, STs catalyze stereo and regiospecific sialylation of distinct glycan acceptors, suggesting that their engineering may yield sialoglycan binding proteins (SiaBP) recognizing both the sialic acid and the acceptor substrate. Thus, SiaBPs may have unique binding specificity that is not recapitulated by traditional lectins or engineered glycosidases. To test this concept, in Aim 1, we perform protein engineering on three different human sialyltransferases to generate three SiaBPs that recognize specific carbohydrate epitopes with high affinity and specificity. These include: ST3Gal-I mutants to recognize Neu5Ac(2-3)Gal(β1-3)GalNAcα; ST6Gal-I mutants to recognize Neu5Ac(2-6)Gal(β1-4)GlcNAcβ; and ST8Sia3 mutants to engage poly sialic acids. We will model the ligand-bound enzyme structures through computational docking and rationally design the mutations to improve binding specificity. We will also perform molecular dynamics (MD) simulation of apo-enzymes and analyze the simulated structures to identify low frequency, collective motions (principal component analysis). The analysis will enable us to introduce mutations to bias the enzyme conformation to one that favors product binding. The rationally designed mutants will be further refined using directed evolution. In Aim 2, purified SiaBPs will be characterized using glycan arrays that bear various sialoglycans. We will additionally assay the binding of SiaBPs to isogenic HEK293T clones and CRISPR-Cas9 KO cell libraries that either contain or have deletions of specific glycoTs. These in cellulo assays complement the glycan arrays and provide a biological context where the engineered proteins will ultimately be used. Successful completion of the project will also result in a platform strategy that may be extended to other glycoTs in the Carbohydrate-Active enzyme database (CAZy.org).

大多数人细胞外蛋白通过连接到Asn的N-连接聚糖和连接到Asn的O-连接聚糖进行后修饰。 连接到Ser/Thr的连接聚糖。这种聚糖控制或微调许多生物过程，包括 细胞生长、分化、细胞粘附和信号传导。因此，糖基化的变化也与 与哺乳动物的病理生理过程如肿瘤转移、宿主-病原体识别、炎症 等理解蛋白质-碳水化合物相互作用在人类健康中的作用的一个重要障碍， 疾病是缺乏一种流线型的技术来快速和准确地表征任意细胞中的聚糖， 组织系统。碳水化合物结合凝集素通常用于表征细胞表面聚糖，但糖结合凝集素的活性不高。 天然植物和动物凝集素的结合特异性和亲和力差。也取得了一些成功， 通过工程化例如唾液酸酶来开发新的聚糖结合蛋白（GBP），以便识别 这些试剂通常与含有唾液酸的聚糖结合，但这些试剂通常仅结合末端残基，对聚糖的特异性较低。 聚糖主链。在本提案中，我们描述了一种替代方法，以工程GBP开始， 糖基转移酶（glycoT），特别是唾液酸转移酶（ST）家族。我们假设 对这类酶进行工程改造可以使得能够以高特异性特异性检测较大的聚糖结构。 在这方面，ST催化不同聚糖受体的立体和区域特异性唾液酸化，这表明它们的结构和功能是不同的。 工程化可以产生识别唾液酸和受体的唾液酸聚糖结合蛋白（SiaBP 衬底因此，SiaBP可能具有独特的结合特异性，其不被传统的凝集素或凝集素受体所概括。 工程化糖苷酶。为了验证这一概念，在目标1中，我们对三种不同的人类进行蛋白质工程， 唾液酸转移酶来产生三种SiaBP，其以高亲和力识别特异性碳水化合物表位， 的特异性这些包括：识别Neu 5Ac（β1-3）Gal（β 2-3）GalNAcα的ST 3Gal-I突变体; 识别Neu 5Ac（β 2-6）Gal（β1-4）GlcNAcβ;和ST 8 Sia 3突变体接合聚唾液酸。我们将模型 配体结合的酶结构，并合理设计突变， 改进结合特异性。我们还将进行脱辅基酶的分子动力学（MD）模拟， 分析模拟结构，以识别低频集体运动（主成分分析）。 分析将使我们能够引入突变，使酶构象偏向有利于产物的构象。 约束力合理设计的突变体将使用定向进化进一步改进。在目标2中，纯化的SiaBP 将使用带有各种唾液酸聚糖的聚糖阵列来表征。我们将另外测定 与含有或具有缺失的等基因HEK 293 T克隆和CRISPR-Cas9 KO细胞文库 特定的糖基这些细胞内测定补充了聚糖阵列，并提供了生物学背景， 最终将使用工程蛋白。该项目的成功完成还将产生一个平台 该策略可以扩展到碳水化合物活性酶数据库（CAZy.org）中的其他glycoT。