NeoLect

Interactions between proteins and glycans are an important component of multiple biological functions, including cell signaling, cell migration, immune recognition and interaction with pathogens. However, Glycomics has yet to reach the level of its counterparts, Genomics and Proteomics, due to the difficulties inherent in carbohydrate analysis. Lectins are glycan receptors that bind mono- and oligosaccharides reversibly and with high specificity and they are commonly used for the structural characterization of carbohydrates. Lectins also have biotechnological applications as biomarker since they can detect altered glycosylation states that occur during pathological states. In the last few years, strong effort has been made into the identification of new lectins as well as into the achievement of a deep understanding of their functions and of the precise mechanism of their association with specific ligands. The production of synthetic lectins with new properties and specificities is an entirely new research domain that is creating strong interest. We propose to design and produce neolectins with controlled valency and specificity based on beta-propeller architecture, i.e. the cyclic repetition of 4 to 7 modules or blades of approximately 40 amino acids. Among the few known lectins that adopt this fold, RSL from the bacterium Ralstonia solanacearum is easy to produce and to engineer and displays strong affinity for human fucosylated epitopes of biological interest. Another beta-propeller lectin from fungal origin (PVL from Psathyrella velutina) is of interest due to its ability to weakly bind to sialylated oligosaccharides. Neolectins with controlled valency will be designed from the 6-blade beta-propeller architecture of RSL that is generated by trimeric association of 2-blade peptides, i.e. presenting 6 fucose binding sites. Site directed mutagenesis together with association of repeats at the genetic level will be used in order to create neolectins with modified valency and different topology in term of active binding site positions in the propeller architecture. These neolectins will be used for studying membrane dynamics that is observed upon binding of toxins with lectin activities such as Shiga- or cholera toxins. Clustering of glycosphingolipids and lectin-induced formation of membrane invaginations will be analyzed on cells and giant unilamellar vesicles. A key point will be the determination of the influence of the neolectin valency and architecture on the formation of membrane tubules and the intracellular trafficking of neolectins. Neolectins with controlled specificity will be designed using a combination of molecular modeling, site-directed mutagenesis and targeted evolution, while taking advantage of the modular architecture of the beta-propellers. NeoRSLs will be modified in order to obtain specificity for various fucosylated oligosaccharides, and more particularly towards Lewis X and Lewis Y epitopes that are overexpressed in some cancers. NeoPVLs will be engineered in order to obtain high affinity for sialylated oligosaccharides since sialic acid binding lectins have direct application for the diagnostic of carcinoma. All neolectins will be assayed on a collection of phenotyped human mucins from saliva and microarrayed human tissues. In a second step, neolectins with specificity for altered glycosylation occurring in some cancerisation process will be selected for their possible application as diagnostic tools.