Neolectins : Synthetic lectins with controled valence and specificity for cellular biology and biotechnology – NeoLect

Proteins were purified from natural sources or produced recombinantly in E. coli.

The structure, specificity and affinity of native and recombinant lectins were characterized with combination of crystallography, glycan arrays, thermal shift assay, surface plasmon resonance and titration microcalorimetry. Giant unilamellar vesicles were used for characterizing the capacity of the lectin to induce invagination by altering the dynamics of glycolipids.

Neolectins were designed based on a beta-propeller scaffold consisting of the cyclic repetition of 6 peptide modules or blades. Molecular modeling has be extensively used for defining hot spots that can influence specificity for different oligosaccharides. Directed mutagenesis was used for designing a mutant with altered valency. Molecular biology was used to create a new object with six blades in one peptide attached by linker. This was the basis for the design of new series of neolectins with controlled number and position of binding sites. Structures were determined.

Soluble lectins with b-propeller fold have been identified in opportunistic bacteria and fungi. The proteins were produced recombinantly and the structure and specificity were fully characterized. The capacity of the fungal lectin to specifically label truncated N-glycans presenting GlcNAc was demonstrated, as well as its ability to specifically labeled tumor cells

The lectin from Ralstonia solanacearum (RSL) has been modified by engineering in order to obtain a neoRSL with controlled valency. Several mutants have been designed, based on structural knowledge of amino acids involved in fucose binding. Among the 9 mutants that have been designed, only three could be produced in soluble form in E. coli. Characterisation of these mutants demonstrated their very strong stability. ITC study confirmed that the mutations reduced the valency from 6 to 3, without affecting affinity of remaining sites for alpha-methyl-fucoside. The use of giant unilamellar vesicles (GUV) decorated with fucose demonstrated that the lectin and the mutants bind to the surface, but only the native lectin with valency 6 is able to induce membrane invagination. Assays on HeLA cells, the mutants have a much slower kinetics of endocytosis, demonstrating the role of valency in internalization.

The neolectins designed with 6 blades that could be individually modified were used for building a library of 13 different proteins with controlled number and position of binding sites. It coudl be demonstrated that avidity effect depends only on the number of binding site, with significant effect observed even with only 2 sites. On the opposite, the capacity to invaginate membrane depends on the distances between the sites.

Our goals have been fullfilled for utilizing lectins as cancer markors and also for developping engineered lectins with controlled valency. The applications in term of understanding membrane dynamics and also bacterial infection have been of high interest. We are still in the process of controlling the specificity of the lectin. Directed evolution by phage display has revealed to be a long and difficult process, but we hope be able to control the specificity of our neolectins in the next few months.

6 publications

1. Audfray, A.; ….. ; Le Pendu, J.; Römer, W.; Varrot, A.; Imberty, A. The fucose-binding lectin from opportunistic pathogen Burkholderia ambifaria binds to both plant and human oligosaccharidic epitopes. J. Biol. Chem. 2012, 287, 4335-4347.
2. Topin, J.; Arnaud, J.; Sarkar, A.; Audfray, A.; ….; Imberty, A.; Thomas, A. Deciphering the glycan preference of bacterial lectins by glycan array and molecular docking with validation by microcalorimetry and crystallography. PLoS ONE. 2013, 8, e71149
3. Audfray A., Varrot A. & Imberty A. Bacteria love our sugars: Interaction between soluble lectins and human fucosylated glycans, structures, thermodynamics and design of competing glycocompounds. C R Chimie 2013, 16, 482-490,
4. Arnaud J., Audfray A. & Imberty A. Binding sugars: from natural lectins to synthetic receptors and engineered neolectins. Chem. Soc. Rev. 2013, 42, 4798-4813,
5. Arnaud, J.; … Römer, W.; Imberty, A.; Audfray, A. Reduction of lectin valency drastically changes glycolipid dynamics in membranes, but not surface avidity. ACS Chem. Biol. 2013, 8, 1918-1924.
6. Arnaud, J.; Tröndle, K.; Claudinon, J.; Audfray, A.; Varrot, A.; Römer, W.; Imberty, A. Membrane deformation by neolectins with engineered glycolipid binding sites. Angew. Chem. Int. Ed. 2014, 53, 9267–9270.
7. Eierhoff, T.; Bastian, B.; Thuenauer, R.; Madl, J.; Audfray, A.; Aigal, S.; Juillot, S.; Rydell, G. E.; Müller, S.; de Bentzmann, S.; Imberty, A.; Fleck, C.; Römer, W. A lipid zipper triggers bacterial invasion. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 12895-12900.
8. Audfray, A.; …Le Pendu , J.; Busser, B.; Imberty, A. A Recombinant Fungal Lectin for Labeling Truncated N-Glycans on Human Cancer Cells, PLOS One, (Accepted with minor modifications).

1 patent
Nouvelles lectines et applications pour la détection de marqueurs d’états pathologiques. A. Imberty, A. Audfray & J. Arnaud. Demande PCT en cours.

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.