Glycoprobes for capture and identification of lectins from biological sources
- Autores: Sira Defaus Fornaguera
- Directores de la Tesis: David Andreu Martínez (dir. tes.), Jordi Robles Brau (dir. tes.), Ricardo Gutiérrez Gallego (dir. tes.)
- Lectura: En la Universitat de Barcelona ( España ) en 2014
- Idioma: inglés
- Tribunal Calificador de la Tesis: Ernest Giralt Lledó (presid.), Eliandre de Oliveira Cacheado (secret.), Manuel Avilés Sánchez (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX Dipòsit Digital de la UB
- Resumen
Glycosylation is a posttranslational modification affecting the fast majority of cellular surfaces through glycolipids and glycoproteins. It has been estimated that over 80% of all proteins are glycosylated and that the glycans decorating the periphery of these molecules are fundamental for the biological function of the entity. Many of these functions have yet to be unraveled. As the modification is a secondary-gene event, it depends on the availability and activity of a set of glycosyltransferases, the availability of both donor and acceptor substrate (both in time and place), as well as many other factors that may include temperature, pH, etc. As a consequence a single glycosylation site may express a certain number of different glycans, i.e., structural microheterogeneity. The importance of glycosylation as primary mediators of cellular communication, protein-protein cross-talk, and even cell-pathogen interaction has been attributed precisely to this phenomenon. In the past few years substantial advancements have been made in the understanding of the function of particular glycosidic epitopes, such as the most relevant blood group antigens ABO, and Sda, (sialyl)Lewisx,y,a,b, the HNK-1 antigen, etc., but many more structures have been identified without an attributed functionality, either as a stand-alone epitope or in conjunction with the underlying peptide structure. Similarly, a serious lack of knowledge still exists on the carbohydrate recognizing molecules, i.e. lectins and their function; even so they have been recognized over the last decades as decisive players in numerous biological processes, ranging from cell-cell communication, fertilization, pathogen-cell adhesion to metastasis. Both deficiencies are directly related to the absence of proper tools to elaborate well-defined carbohydrate epitopes for the study of their interaction characteristics and their employment in the discovery of new complementary molecules. Consequently, there is an increasing interest in finding powerful and nanosized tools to screen for these molecules and to study their carbohydrate interactions in detail. In this Thesis, two complementary approaches are described to characterize lectin-carbohydrate interactions with high sensitivity, low sample consumption, and without the need for sample labeling: SPR and CREDEX-MS. In SPR, we have developed an approach where the sugar is immobilized onto a sensor surface through a tailor-made peptide module that allows (1) to capture the lectin, (2) to characterize the interaction through kinetic and thermodynamic parameters, and (3) to identify the interacted protein by mass spectrometry. In CREDEX-MS, based on proteolytic excision of protein-carbohydrate complexes and mass spectrometric analysis, the peptides conforming the carbohydrate binding domain are identified. After completing a preliminary phase where the above mentioned methodologies are optimized and used for the study of carbohydrate-protein interactions using purified well-known lectins; the established platforms are employed to analyze carbohydrate-driven interactions in more complex systems. Specifically, SPR and CREDEX-MS techniques are applied in a research project on molecular aspects of reproduction with the main objective of disclosing the molecular elements that participate in the first steps of fertilization in bovine species, namely: formation of the sperm reservoir in the oviductal epithelium and gamete recognition (oocyte (ZP)-sperm interaction).
