Structure and engineering of tandem repeat lectins

International audience(100 − 120 words) Through their ability to bind complex glycoconjugates, lectins have unique specificity and potential for biomedical and biotechnological applications. In particular, lectins with short repeated peptides forming carbohydrate-binding domains are not only of high interest for understanding protein evolution but can also be used as scaffold for engineering novel receptors. Synthetic glycobiology now provides the tools for engineering the specificity of lectins as well as their structure, multivalency and topologies. This review focuses on the structure and diversity of two families of tandem-repeat lectins, i.e. β-trefoils and β-propellers, demonstrated as the most promising scaffold for engineering novel lectins

Ingénierie de neo-lectines et lectines Janus

Glycobiology is a rapidly growing field of natural sciences with a focus on glycans, glycoconjugates and glycan binding proteins. Lectins are sugar-binding proteins present in all types of organisms and they display wide range of biological functions. As encoded in their name (from Latin - legere – to select), each lectin is specific only to a finite group of glycans and in order to ensure higher affinity, they are generally multivalent. Lectins are powerful glycan-profiling tools and even though some of them already found their applications, the discovery of novel lectins is still desirable. In this regard, involvement of synthetic biology and protein engineering are of high interest for building of lectin architecture and tuning their specificity.Various approaches for lectin discovery or engineering are presented in this thesis. The thesis is composed of several chapters, where the introduction is dedicated to the general description of lectins and lectin engineering with the respect to their specificity and topology, including a short review on engineering of β-propeller and β-trefoil lectins. The results are presented in three scientific articles (in the format of preprint or manuscripts in preparation). The first publication describes the discovery and characterization of novel pore-forming lectin with specificity toward cancer cells glyco-epitope. In the second manuscript, synthetic biology approach was used to create artificial proteins with the ability to recognize plant cell wall polymers and to be used as glue proteins in the construction of an artificial plant cell wall. The third manuscript generalizes the Janus lectin strategy as a universal tool for creation of bispecific chimeras with increased valency. The last chapter summarizes the achieved results and propose new perspectives and challenges giving the special importance to the continuation of lectin engineering.La glycobiologie est un domaine des sciences naturelles en plein essor qui se concentre sur les glycanes, les glycoconjugués et les protéines de liaison aux glycanes. Les lectines sont des protéines de reconnaissance des sucres présentes dans tous les types d'organismes et elles présentent un large éventail de fonctions biologiques. Comme indiqué dans leur nom (du latin - legere - sélectionner), chaque lectines est spécifique seulement d’un groupe limité de glycanes et, afin d'assurer une affinité plus élevée, elles sont généralement multivalentes. Les lectines sont de puissants outils de profilage des glycanes et même si certaines d'entre elles ont déjà trouvé leurs applications, la découverte de nouvelles lectines est toujours souhaitable. Dans cette optique, l'implication de la biologie synthétique et de l'ingénierie des protéines est d'un grand intérêt pour la modification de l'architecture ou même de la spécificité des lectines.Différentes approches pour la découverte ou l'ingénierie des lectines sont présentées dans cette thèse. La thèse est composée de plusieurs chapitres, où l'introduction est dédiée à la description générale des lectines et de leur ingénierie pour ce qui concerne leur spécificité et leur topologie. Elle comprend une brève revue de l'ingénierie des lectines β-propeller et β-trefoil. Les résultats sont présentés dans trois articles scientifiques (préprint ou manuscrit en préparation). La première publication décrit la découverte et la caractérisation d'une nouvelle lectine formant des pores dans les membranes et présentant une spécificité pour des glyco-epitopes de cellules cancéreuses. Dans le deuxième manuscrit, une approche de biologie synthétique a été utilisée pour créer des protéines artificielles ayant la capacité de reconnaître les polysaccharides de la paroi cellulaire végétale et ainsi les utiliser comme glue dans la construction de paroi cellulaire végétale artificielle. Le troisième manuscrit généralise la stratégie des lectines Janus comme un outil universel pour la création de chimères bispécifiques avec une valence accrue. Le dernier chapitre résume les résultats obtenus et propose de nouvelles perspectives et de nouveaux défis en accordant une importance particulière à la poursuite de l'ingénierie des lectines

Engineering of neo-lectins and janus lectins

La glycobiologie est un domaine des sciences naturelles en plein essor qui se concentre sur les glycanes, les glycoconjugués et les protéines de liaison aux glycanes. Les lectines sont des protéines de reconnaissance des sucres présentes dans tous les types d'organismes et elles présentent un large éventail de fonctions biologiques. Comme indiqué dans leur nom (du latin - legere - sélectionner), chaque lectines est spécifique seulement d’un groupe limité de glycanes et, afin d'assurer une affinité plus élevée, elles sont généralement multivalentes. Les lectines sont de puissants outils de profilage des glycanes et même si certaines d'entre elles ont déjà trouvé leurs applications, la découverte de nouvelles lectines est toujours souhaitable. Dans cette optique, l'implication de la biologie synthétique et de l'ingénierie des protéines est d'un grand intérêt pour la modification de l'architecture ou même de la spécificité des lectines.Différentes approches pour la découverte ou l'ingénierie des lectines sont présentées dans cette thèse. La thèse est composée de plusieurs chapitres, où l'introduction est dédiée à la description générale des lectines et de leur ingénierie pour ce qui concerne leur spécificité et leur topologie. Elle comprend une brève revue de l'ingénierie des lectines β-propeller et β-trefoil. Les résultats sont présentés dans trois articles scientifiques (préprint ou manuscrit en préparation). La première publication décrit la découverte et la caractérisation d'une nouvelle lectine formant des pores dans les membranes et présentant une spécificité pour des glyco-epitopes de cellules cancéreuses. Dans le deuxième manuscrit, une approche de biologie synthétique a été utilisée pour créer des protéines artificielles ayant la capacité de reconnaître les polysaccharides de la paroi cellulaire végétale et ainsi les utiliser comme glue dans la construction de paroi cellulaire végétale artificielle. Le troisième manuscrit généralise la stratégie des lectines Janus comme un outil universel pour la création de chimères bispécifiques avec une valence accrue. Le dernier chapitre résume les résultats obtenus et propose de nouvelles perspectives et de nouveaux défis en accordant une importance particulière à la poursuite de l'ingénierie des lectines.Glycobiology is a rapidly growing field of natural sciences with a focus on glycans, glycoconjugates and glycan binding proteins. Lectins are sugar-binding proteins present in all types of organisms and they display wide range of biological functions. As encoded in their name (from Latin - legere – to select), each lectin is specific only to a finite group of glycans and in order to ensure higher affinity, they are generally multivalent. Lectins are powerful glycan-profiling tools and even though some of them already found their applications, the discovery of novel lectins is still desirable. In this regard, involvement of synthetic biology and protein engineering are of high interest for building of lectin architecture and tuning their specificity.Various approaches for lectin discovery or engineering are presented in this thesis. The thesis is composed of several chapters, where the introduction is dedicated to the general description of lectins and lectin engineering with the respect to their specificity and topology, including a short review on engineering of β-propeller and β-trefoil lectins. The results are presented in three scientific articles (in the format of preprint or manuscripts in preparation). The first publication describes the discovery and characterization of novel pore-forming lectin with specificity toward cancer cells glyco-epitope. In the second manuscript, synthetic biology approach was used to create artificial proteins with the ability to recognize plant cell wall polymers and to be used as glue proteins in the construction of an artificial plant cell wall. The third manuscript generalizes the Janus lectin strategy as a universal tool for creation of bispecific chimeras with increased valency. The last chapter summarizes the achieved results and propose new perspectives and challenges giving the special importance to the continuation of lectin engineering

Ingénierie de neo-lectines et lectines Janus

Glycobiology is a rapidly growing field of natural sciences with a focus on glycans, glycoconjugates and glycan binding proteins. Lectins are sugar-binding proteins present in all types of organisms and they display wide range of biological functions. As encoded in their name (from Latin - legere – to select), each lectin is specific only to a finite group of glycans and in order to ensure higher affinity, they are generally multivalent. Lectins are powerful glycan-profiling tools and even though some of them already found their applications, the discovery of novel lectins is still desirable. In this regard, involvement of synthetic biology and protein engineering are of high interest for building of lectin architecture and tuning their specificity.Various approaches for lectin discovery or engineering are presented in this thesis. The thesis is composed of several chapters, where the introduction is dedicated to the general description of lectins and lectin engineering with the respect to their specificity and topology, including a short review on engineering of β-propeller and β-trefoil lectins. The results are presented in three scientific articles (in the format of preprint or manuscripts in preparation). The first publication describes the discovery and characterization of novel pore-forming lectin with specificity toward cancer cells glyco-epitope. In the second manuscript, synthetic biology approach was used to create artificial proteins with the ability to recognize plant cell wall polymers and to be used as glue proteins in the construction of an artificial plant cell wall. The third manuscript generalizes the Janus lectin strategy as a universal tool for creation of bispecific chimeras with increased valency. The last chapter summarizes the achieved results and propose new perspectives and challenges giving the special importance to the continuation of lectin engineering.La glycobiologie est un domaine des sciences naturelles en plein essor qui se concentre sur les glycanes, les glycoconjugués et les protéines de liaison aux glycanes. Les lectines sont des protéines de reconnaissance des sucres présentes dans tous les types d'organismes et elles présentent un large éventail de fonctions biologiques. Comme indiqué dans leur nom (du latin - legere - sélectionner), chaque lectines est spécifique seulement d’un groupe limité de glycanes et, afin d'assurer une affinité plus élevée, elles sont généralement multivalentes. Les lectines sont de puissants outils de profilage des glycanes et même si certaines d'entre elles ont déjà trouvé leurs applications, la découverte de nouvelles lectines est toujours souhaitable. Dans cette optique, l'implication de la biologie synthétique et de l'ingénierie des protéines est d'un grand intérêt pour la modification de l'architecture ou même de la spécificité des lectines.Différentes approches pour la découverte ou l'ingénierie des lectines sont présentées dans cette thèse. La thèse est composée de plusieurs chapitres, où l'introduction est dédiée à la description générale des lectines et de leur ingénierie pour ce qui concerne leur spécificité et leur topologie. Elle comprend une brève revue de l'ingénierie des lectines β-propeller et β-trefoil. Les résultats sont présentés dans trois articles scientifiques (préprint ou manuscrit en préparation). La première publication décrit la découverte et la caractérisation d'une nouvelle lectine formant des pores dans les membranes et présentant une spécificité pour des glyco-epitopes de cellules cancéreuses. Dans le deuxième manuscrit, une approche de biologie synthétique a été utilisée pour créer des protéines artificielles ayant la capacité de reconnaître les polysaccharides de la paroi cellulaire végétale et ainsi les utiliser comme glue dans la construction de paroi cellulaire végétale artificielle. Le troisième manuscrit généralise la stratégie des lectines Janus comme un outil universel pour la création de chimères bispécifiques avec une valence accrue. Le dernier chapitre résume les résultats obtenus et propose de nouvelles perspectives et de nouveaux défis en accordant une importance particulière à la poursuite de l'ingénierie des lectines

Tuning specificity and topology of lectins through synthetic biology

International audienceLectins are non-immunoglobulin and non-catalytic glycan binding proteins that are able to decipher the structure and function of complex glycans. They are widely used as biomarkers for following alteration of glycosylation state in many diseases and have application in therapeutics. Controlling and extending lectin specificity and topology is the key for obtaining better tools. Furthermore, lectins and other glycan binding proteins can be combined with additional domains, providing novel functionalities. We provide a view on the current strategy with a focus on synthetic biology approaches yielding to novel specificity, but other novel architectures with novel application in biotechnology or therapy

The Two Sweet Sides of Janus Lectin Drive Crosslinking of Liposomes to Cancer Cells and Material Uptake

International audienceA chimeric, bispecific Janus lectin has recently been engineered with different, rationally oriented recognition sites. It can bind simultaneously to sialylated and fucosylated glycoconjugates. Because of its multivalent architecture, this lectin reaches nanomolar avidities for sialic acid and fucose. The lectin was designed to detect hypersialylation—a dysregulation in physiological glycosylation patterns, which promotes the tumor growth and progression of several cancer types. In this study, the characteristic properties of this bispecific Janus lectin were investigated on human cells by flow cytometry and confocal microscopy in order to understand the fundamentals of its interactions. We evaluated its potential in targeted drug delivery, precisely leading to the cellular uptake of liposomal content in human epithelial cancer cells. We successfully demonstrated that Janus lectin mediates crosslinking of glyco-decorated giant unilamellar vesicles (GUVs) and H1299 lung epithelial cells. Strikingly, the Janus lectin induced the internalization of liposomal lipids and also of complete GUVs. Our findings serve as a solid proof of concept for lectin-mediated targeted drug delivery using glyco-decorated liposomes as possible drug carriers to cells of interest. The use of Janus lectin for tumor recognition certainly broadens the possibilities for engineering diverse tailor-made lectin constructs, specifically targeting extracellular structures of high significance in pathological conditions

Extending Janus lectins architecture: Characterization and application to protocells

International audienceSynthetic biology is a rapidly growing field with applications in biotechnology and biomedicine. Through various approaches, remarkable achievements, such as cell and tissue engineering, have been already accomplished. In synthetic glycobiology, the engineering of glycan binding proteins is being exploited for producing tools with precise topology and specificity. We developed the concept of engineered chimeric lectins, i.e., Janus lectin, with increased valency, and additional specificity. The novel engineered lectin, assembled as a fusion protein between the b-propeller domain from Ralstonia solanacearum and the b-trefoil domain from fungus Marasmius oreades, is specific for fucose and a-galactose and its unique protein architecture allows to bind these ligands simultaneously. The protein activity was tested with glycosylated giant unilamellar vesicles, resulting in the formation of proto-tissue-like structures through cross-linking of such protocells. The engineered protein recognizes and binds H1299 human lung epithelial cancer cells by its two domains. The biophysical properties of this new construct were compared with the two already existing Janus lectins, RSL-CBM40 and RSL-CBM77 Rf. Denaturation profiles of the proteins indicate that the fold of each has a significant role in protein stability and should be considered during protein engineering

Building an Artificial Plant Cell Wall on a Lipid Bilayer by Assembling Polysaccharides and Engineered Proteins

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Immobilization of Biantennary N-Glycans Leads to Branch Specific Epitope Recognition by LSECtin

International audienceThe molecular recognition features of LSECtin toward asymmetric N-glycans have been scrutinized by NMR and compared to those occurring in glycan microarrays. A pair of positional glycan isomers (LDN3 and LDN6), a nonelongated GlcNAc4Man3 N-glycan (G0), and the minimum binding epitope (the GlcNAcβ1-2Man disaccharide) have been used to shed light on the preferred binding modes under both experimental conditions. Strikingly, both asymmetric LDN3 and LDN6 N-glycans are recognized by LSECtin with similar affinities in solution, in sharp contrast to the results obtained when those glycans are presented on microarrays, where only LDN6 was efficiently recognized by the lectin. Thus, different results can be obtained using different experimental approaches, pointing out the tremendous difficulty of translating in vitro results to the in vivo environment

Immobilization of Biantennary N-Glycans Leads to Branch Specific Epitope Recognition by LSECtin

International audienceThe molecular recognition features of LSECtin toward asymmetric N-glycans have been scrutinized by NMR and compared to those occurring in glycan microarrays. A pair of positional glycan isomers (LDN3 and LDN6), a nonelongated GlcNAc4Man3 N-glycan (G0), and the minimum binding epitope (the GlcNAcβ1-2Man disaccharide) have been used to shed light on the preferred binding modes under both experimental conditions. Strikingly, both asymmetric LDN3 and LDN6 N-glycans are recognized by LSECtin with similar affinities in solution, in sharp contrast to the results obtained when those glycans are presented on microarrays, where only LDN6 was efficiently recognized by the lectin. Thus, different results can be obtained using different experimental approaches, pointing out the tremendous difficulty of translating in vitro results to the in vivo environment