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).

大多数人类细胞外蛋白是通过附着在ASN上的N-连接的聚糖和O-进行的转化后修饰的 连接到Ser/Thr的链接的聚糖。这样的聚糖控制或微调许多生物学过程，包括 细胞生长，分化，细胞粘合剂和信号传导。结果，糖基化的变化也是相关的 随着哺乳动物的病理生理过程，例如肿瘤转移，宿主 - 病原体识别，炎症 等等。了解蛋白质 - 碳水化合物相互作用在人类健康和 疾病是缺乏精简的技术来快速，准确地表征任意细胞中的聚糖/ 组织系统。碳水化合物结合讲座通常用于表征细胞表面糖，但是 天然植物和动物讲座的结合特异性和亲和力很差。也有一些成功 例如，通过工程酶开发新型的聚糖结合蛋白（GBP），以识别 含有聚糖的唾液酸，但是这些试剂通常仅结合末端，保留的特异性较少 聚糖骨干。在此提案中，我们描述了一种从事工程Gbps的替代方法 糖基转移酶（Glycot），尤其是重点是辅助转移酶（ST）家族。我们假设这一点 工程这类酶可以使具有高特异性的较大的聚糖结构进行特定检测。 在这方面，STS催化了不同聚糖受体的立体声和调节性诊断，表明它们 工程可能会产生唾液酸结合蛋白（SIABP），以识别唾液酸和受体 基材。那就是，Siabps可能具有独特的约束性特异性，而传统讲座或 设计的糖苷酶。为了测试这个概念，在AIM 1中，我们对三个不同的人进行蛋白质工程 锡基转移酶产生三个siabps，识别具有高亲和力和 特异性。其中包括：ST3GAL-I突变体以识别neu5ac（2-3）gal（β1-3）GalNACα； st6gal-i突变体 识别neu5ac（2-6）gal（β1-4）GlcNACβ；和ST8SIA3突变体可以接合多氨酸。我们将建模 通过计算对接和合理设计突变的配体结合的酶结构 提高结合特异性。我们还将对Apo-酶和 分析模拟结构以识别低频，集体运动（主成分分析）。 该分析将使我们能够引入突变以使酶考虑偏向于偏爱产品的突变。 结合。理性设计的突变体将使用定向进化进一步完善。在AIM 2中，纯化的siabps 将使用带有各种唾液聚糖的聚糖阵列来表征。我们还将主张绑定 siabps of Inogenic HEK293T克隆和CRISPR-CAS9 KO细胞库，其中包含或具有删除 特定的聚糖。这些在纤维素测定中完成了聚糖阵列，并提供了一个生物环境 最终将使用工程蛋白质。成功完成项目也将导致平台 可以扩展到碳水化合物活性酶数据库（cazy.org）中其他聚糖的策略。