Identification of cross-linking pattern in human elastin

弹性蛋白是脊椎动物细胞外基质的必需蛋白质，具有独特的性质，如弹性，弹性和极端的耐久性，这使得它对不同组织和器官如主动脉，肺，皮肤或软骨的长期功能至关重要。生物聚合物由其前体原弹性蛋白的单元组成，这些单元通过赖氨酸残基连接并形成各种多官能交联。由于其高疏水性和复杂的交联模式，弹性蛋白是完全不溶的，并且对酶降解非常耐受，这限制了结构研究。因此，实际上对不同人体组织中弹性蛋白的交联模式一无所知。然而，在分子水平上研究弹性蛋白的结构以更好地理解其在健康组织中的功能以及与心血管疾病如动脉粥样硬化、肺气肿和主动脉瓣狭窄沿着的结构变化是非常重要的。此外，对弹性纤维结构的详细了解将有助于开发基于蛋白质的生物材料，这些材料可用于各种医疗应用。因此，本项目的目的是开发和应用分析和生物信息学方法来表征从人主动脉分离的弹性蛋白的交联模式。基于不同的模型系统，如交联弹性蛋白肽，交联弹性蛋白多肽和交联弹性蛋白原质谱方法将被开发，这使得能够识别和测序弹性蛋白的交联特征。然后，使用特定弹性蛋白酶对分离的弹性蛋白进行受控降解并随后鉴定交联肽将允许确定分子间和分子内交联的确切位置以及参与交联的原弹性蛋白分子的结构域。该项目将为弹性蛋白的结构提供全面而重要的见解。总的来说，了解弹性蛋白的结构将支持针对弹性组织疾病的定向治疗的发展，包括酶抑制剂，伤口再生材料或血管假体。

Elastin is an essential protein of the extracellular matrix of vertebrates and possesses unique properties such as elasticity, resilience and extreme durability, which make it crucial for the long-term function of different tissues and organs such as aorta, lung, skin or cartilage. The biopolymer is composed of units of its precursor tropoelastin, which are connected via lysine residues and form a variety of polyfunctional cross-links. Due to its high hydrophobicity and complex cross-linking pattern elastin is completely insoluble and very resistant to enzymatic degradation, which limits structure investigations. Hence, virtually nothing is known about the cross-linking pattern of elastin in different human tissues. It is, however, of major importance to investigate the structure of elastin on the molecular level to better understand its functions in healthy tissues as well as structural changes that go along with cardiovascular diseases such as atherosclerosis, lung emphysema and aortic stenosis. Moreover, detailed insights into the structure of elastic fibers will aid in the development of protein-based biomaterials, which may be used for a variety of medical applications. Therefore, the aim of this project is the development and the application of analytical and bioinformatics methods to characterize the cross-linking pattern of elastin isolated from human aorta. Based on different model systems such as cross-linked elastin peptides, cross-linked elastin polypeptides and cross-linked tropoelastin mass spectrometric methods will be developed, which enable identifying and sequencing cross-links characteristic of elastin. Controlled degradation of isolated elastin using specific elastases and subsequent identification of cross-linked peptides will then allow to determine the exact positions of inter- and intramolecular cross-links as well as the domains of tropoelastin molecules involved in cross-linking. The project will provide a comprehensive and significant insight into the structure of elastin. Overall, understanding the structure of elastin will support the development of directed therapies against diseases of elastic tissues, including enzyme inhibitors, wound regeneration materials or vascular prostheses.