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.
A 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.
The cell wall constitutes a fundamental structural component of plant cells, providing them with mechanical resistance and flexibility. Mimicking that wall is a critical step in the conception of an experimental model of the plant cell. The assembly of cellulose/hemicellulose in the form of cellulose nanocrystals and xyloglucans as a representative model of the plant cell wall has already been mastered, however, those models lacked the pectin component. In this work, we used an engineered chimeric protein designed for bridging pectin to the cellulose/hemicellulose network, therefore achieving the assembly of complete cell wall mimics. We first engineered proteins, i.e. carbohydrate-binding module from Ruminococcus flavefaciens able to bind oligo-galactorunan, resulting in high-affinity polygalacturonan receptors with Kd in the micromolar range. A Janus protein, with cell wall gluing property, was then designed by assembling this CBM with a Ralstonia solanacearum lectin specific for fucosylated xyloglucans. The resulting supramolecular architecture is able to bind fucose-containing xyloglucans and homogalacturonan ensuring high affinity for both. A two-dimension assembly of an artificial plant cell wall was then built first on synthetic polymer and then on supported lipid bilayer. Such artificial cell wall can serve as a basis for the development of plant cell mechanical models and thus deepen the understanding of the principles underlying various aspects of plant cells and tissues.
The 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.
Lectins are efficient multivalent glycan receptors, deciphering the glyco-code on cell surfaces. The β-trefoil fold, characterized by three lobe-shaped repeats, is adopted by several classes of lectins, often associated with other domains having enzymatic or toxic activity. Based on the UniLectin3D database classification, the sequence signature of trefoil lobes was defined and used to predict 44714 lectins from 4497 species. Among them, SaroL-1 from the lower eukaryote Salpingoeca rosetta was predicted to contain both β-trefoil and aerolysin-like pore-forming domain. Recombinant SaroL-1 binds to galactose and derivatives, with a stronger affinity for cancer-related α-galactosylated epitopes such as glycosphingolipid Gb3 embedded in giant unilamellar vesicles or cell membranes. Crystal structures in complex with Gb3 trisaccharide and GalNAc show similarity with pore-forming toxins. Recognition of the αGal epitope on glycolipids was necessary for hemolysis of rabbit erythrocytes and toxicity on cancer cells through carbohydrate-dependent pore-formation.
Choanoflagellates are primitive protozoa used as models for animal evolution. They express a large variety of multi-domain proteins contributing to adhesion and cell communication, thereby providing a rich repertoire of molecules for biotechnology. Adhesion often involves proteins adopting a β-trefoil fold with carbohydrate-binding properties therefore classified as lectins. Sequence database screening with a dedicated method resulted in TrefLec, a database of 44714 β-trefoil candidate lectins across 4497 species. TrefLec was searched for original domain combinations, which led to single out SaroL-1 in the choanoflagellate Salpingoeca rosetta, that contains both β-trefoil and aerolysin-like pore-forming domains. Recombinant SaroL-1 is shown to bind galactose and derivatives, with a stronger affinity for cancer-related α-galactosylated epitopes such as the glycosphingolipid Gb3, when embedded in giant unilamellar vesicles or cell membranes. Crystal structures of complexes with Gb3 trisaccharide and GalNAc provided the basis for building a model of the oligomeric pore. Finally, recognition of the αGal epitope on glycolipids required for hemolysis of rabbit erythrocytes suggests that toxicity on cancer cells is achieved through carbohydrate-dependent pore-formation.
The cell wall constitutes a fundamental structural component of plant cells, providing them with mechanical resistance and flexibility. Mimicking this wall is a critical step in the conception of an experimental model of the plant cell. The assembly of cellulose/hemicellulose in the form of cellulose nanocrystals and xyloglucans as a representative model of the plant cell wall has already been mastered; however, these models lacked the pectin component. In this work, we used an engineered chimeric protein designed for bridging pectin to the cellulose/hemicellulose network, therefore achieving the assembly of complete cell wall mimics. We first engineered a carbohydrate-binding module from Ruminococcus flavefaciens able to bind oligogalacturonan, resulting in high-affinity polygalacturonan receptors with K d in the micromolar range. A Janus protein, with cell wall gluing property, was then designed by assembling this carbohydrate-binding module with a Ralstonia solanacearum lectin specific for fucosylated xyloglucans. The resulting supramolecular architecture is able to bind fucose-containing xyloglucans and homogalacturonan, ensuring high affinity for both. A two-dimensional assembly of an artificial plant cell wall was then built first on synthetic polymer and then on the supported lipid bilayer. Such an artificial cell wall can serve as a basis for the development of plant cell mechanical models and thus deepen the understanding of the principles underlying various aspects of plant cells and tissues.
Synthetic 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 developed for producing tools with precise topology and specificity. We developed the concept of chimeric lectins, i.e., Janus lectin, with increased valency, and additional specificity. The novel engineered lectin, assembled as a fusion protein between the β-propeller domain from Ralstonia solanacearum and the β-trefoil domain from fungus Marasmius oreades, is specific for fucose and α-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 synthetic protein binds to H1299 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-CBM77Rf. 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.
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