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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的人类上皮组织是重要的生物屏障，可分泌一种称为粘液的独特水凝胶。组织产生不同类型的粘液，提供特定的生物功能，如水合、病原体防御，并介导物质向细胞表面的运动。粘液的主要成分，粘蛋白对于凝胶结构和功能至关重要。粘蛋白是一个由 20 多种蛋白质组成的多样化家族由富含丝氨酸/苏氨酸的大的棒状结构域与附着的糖或聚糖组成。分子级粘液由于组织和物种特异性的异质聚糖模式，研究一直具有挑战性，如以及不同的蛋白质表达水平和剪接导致不同长度和序列的结构。粘蛋白表达、剪接和糖基化的错误调节会导致结构改变，从而可能影响具有与感染、炎症和癌症相关的生物学功能。研究人员通常利用从农场动物来源中分离出粘蛋白用于此类研究，但该来源存在批次间差异，未化学定义且具有无法系统地分析的非人类聚糖模式的结构改变了。目前，对可以在分子水平上进行调整的化学定义的粘蛋白的需求尚未得到满足并具有人类糖基化模式。这些材料对于探索这些重要的作用至关重要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制备合成人类粘蛋白，用于探测聚糖-病原体相互作用。技巧来自碳水化合物化学、氨基酸 N-羧酸酐 (NCA) 聚合和酶促糖基化，将被结合起来生成具有完全可调特性的材料。我将生成一个面板具有不同长度、氨基酸组成、糖基化的化学定义粘蛋白糖多肽密度和聚糖结构。结构设计将以已发表的天然人类糖组分析为指导与气道感染有关的粘蛋白。所有糖多肽都将得到全面的理化表征使用各种光谱、显微镜和生化方法来确定特性。粘蛋白及其聚糖先前已被证明会影响病原微生物的活性，例如粘附、生物膜形成和毒力特征。合成粘蛋白将用于揭示聚糖的呈现如何影响这些致病菌功能。对于天然粘蛋白来说，此类研究是不可能的，因为糖基化无法控制并且是通常甚至没有特征。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。这种跨学科方法将结合多个领域的技术回答有关感染的重要问题，这些问题不能仅通过生物学方法来解决。这所提出的材料可以作为抗菌剂或粘膜替代疗法具有治疗应用。