Chemically Tunable Mucins to Probe Pathogenic Function in the Epithelial Milieu

化学可调节粘蛋白探测上皮环境中的致病功能

• 批准号: 10441134
• 负责人: Victoria Rose Kohout
• 金额: $ 6.76万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10441134
• 关键词: Acetylgalactosamine; Address; Adhesions; Affect; Amino Acids; Animal Sources; Binding; Biochemical; Biocompatible Materials; Biological; Biological Assay; Biological Process; Candida albicans; Carbohydrate Chemistry; Cell surface; Cells; Chemicals; Cues; Cystic Fibrosis; DNA; Disease; Enzyme-Linked Immunosorbent Assay; Enzymes; Epithelial; Family; Fucose; Fucosyltransferase; Gel; Gene Expression; Glycoproteins; Hand; Health; Heterogeneity; Human; Hydration status; Hydrogels; Immune; Incubated; Infection; Inflammation; Lectin; Length; Light; Livestock; Lubrication; Malignant Neoplasms; Mediating; Metabolic; Methods; Microbe; Microbial Biofilms; Microscopic; Microscopy; Modification; Molecular; Molecular Conformation; Molecular Structure; Movement; Mucins; Mucous body substance; Outcome; Pathogenicity; Pathway interactions; Pattern; Peptide Hydrolases; Peptides; Polymers; Polysaccharides; Property; Proteins; Pseudomonas aeruginosa; Publishing; RNA Splicing; Replacement Therapy; Research; Research Personnel; Respiratory Tract Infections; Rod; Role; Salts; Serine; Sialic Acids; Sialyltransferases; Source; Staphylococcus aureus; Structure; Structure-Activity Relationship; Techniques; Therapeutic; Threonine; Tissues; Trisaccharides; Variant; Vertebral column; Virulence; Water; Work; animal tissue; antimicrobial; asthmatic patient; chemical synthesis; density; design; fundamental research; glycosylation; interdisciplinary approach; microbiome; monomer; pathogen; pathogenic microbe; polymerization; protein expression; sugar; trait; unnatural amino acids

Abstract Human epithelial tissues are essential biological barriers that secrete a unique hydrogel known as mucus. Tissues generate distinct types of mucus that provide specific biological functions like hydration, pathogen defense, and mediating the movement substances toward the cell surface. The major component of mucus, mucin proteins, is critical for gel structure and function. Mucins are a diverse family of 20+ proteins characterized by a large, rod-like domain rich serine/threonine with attached saccharides, or glycans. Molecular-level mucus studies have been challenging due to heterogeneous glycan patterns that are tissue and species specific, as well as varied protein expression levels and splicing that result in structures with varied lengths and sequences. Misregulation of mucin expression, splicing, and glycosylation results in altered structures that may affect biological function with outcomes relevant to infection, inflammation, and cancer. Researchers typically utilize mucins isolated from farm animal sources for such studies, but this source suffers from batch-to-batch variation, structures that are not chemically defined and have non-human glycan patterns that cannot be systematically altered. Currently, there is an unmet need for chemically-defined mucins that can be tuned at the molecular level and possess human glycosylation patterns. Such materials are essential to probe the role of these vital biomaterials in health and disease. The proposed research will address this critical need by developing a method to prepare synthetic human mucins, which will be applied to probe glycan-pathogen interactions. Techniques from carbohydrate chemistry, amino acid N-carboxyanhydride (NCA) polymerization, and enzymatic glycosylation, will be combined to generate materials with fully tunable properties. I will generate a panel of chemically-defined mucin glycopolypeptides with varied lengths, amino acid compositions, glycosylation densities, and glycan structures. Structure design will be guided by published glycomic analysis of native human mucins implicated in airway infections. All glycopolypeptides will be fully characterized for physicochemical properties using a variety of spectroscopic, microscopic, and biochemical methods. Mucins and their glycans have previously been shown to affect activity of pathogenic microbes such as adhesion, biofilm formation, and virulence traits. Synthetic mucins will be applied to reveal how glycan presentation affects these pathogenic functions. Such studies are not possible with native mucins since glycosylation cannot be controlled and is typically not even characterized. Overall, I aim to shed light on the molecular structure-function relationship between mucins and microbes. This interdisciplinary approach will combine techniques from multiple fields to answer important questions about infection that cannot be undertaken by biological methods alone. The proposed materials could have therapeutic applications as antimicrobials or in muco-replacement therapies.

摘要 人类上皮组织是分泌称为粘液的独特水凝胶的基本生物屏障。 组织产生不同类型的粘液，提供特定的生物功能，如水合作用，病原体 防御，并介导向细胞表面的运动物质。粘液的主要成分， 粘蛋白是凝胶结构和功能的关键。粘蛋白是一个由20多种蛋白质组成的多样化家族， 通过一个大的、杆状的富含丝氨酸/苏氨酸的结构域，并连接有丝氨酸或聚糖。分子级粘液 由于组织和物种特异性的异质聚糖模式， 以及不同的蛋白质表达水平和剪接，导致具有不同长度和序列的结构。 粘蛋白表达、剪接和糖基化的失调导致结构改变， 生物学功能与感染、炎症和癌症相关的结果。研究人员通常利用 从农场动物源分离的粘蛋白用于这类研究，但这种来源存在批次间差异， 非化学定义的结构，并且具有不能系统分析的非人聚糖模式 改变了目前，对于可以在分子水平上调节的化学定义的粘蛋白存在未满足的需求 并具有人糖基化模式。这些材料对于探索这些重要的 健康和疾病中的生物材料。拟议的研究将通过开发一种方法来解决这一关键需求 以制备合成的人类粘蛋白，其将被应用于探测聚糖-病原体相互作用。技术 从碳水化合物化学，氨基酸N-羧酸酐（NCA）聚合，和酶 糖基化，将组合以产生具有完全可调性质的材料。我将生成一个面板， 具有不同长度、氨基酸组成、糖基化的化学定义的粘蛋白糖多肽 密度和聚糖结构。结构设计将由已发表的天然人的糖组学分析指导。 与呼吸道感染有关的粘蛋白。所有糖多肽都将进行充分的理化表征， 使用各种光谱学、显微镜和生物化学方法测定其性质。粘蛋白及其聚糖 先前已显示影响病原微生物的活性，如粘附、生物膜形成， 毒力性状合成粘蛋白将被应用于揭示聚糖呈递如何影响这些致病性 功能协调发展的用天然粘蛋白进行这样的研究是不可能的，因为糖基化不能被控制， 通常甚至没有特征。总的来说，我的目标是阐明分子结构与功能的关系 粘蛋白和微生物之间的联系这种跨学科的方法将联合收割机技术从多个领域， 回答了关于感染的重要问题，这些问题不能仅仅通过生物学方法来解决。的 所提出的材料可具有作为抗微生物剂或粘膜替代疗法的治疗应用。