Lectin PLL3, a Novel Monomeric Member of the Seven-Bladed ß-Propeller Lectin Family.

The Photorhabdus species is a Gram-negative bacteria of the family Morganellaceae that is known for its mutualistic relationship with Heterorhabditis nematodes and pathogenicity toward insects. This study is focused on the characterization of the recombinant lectin PLL3 with an origin in P. laumondii subsp. laumondii. PLL3 belongs to the PLL family of lectins with a seven-bladed ß-propeller fold. The binding properties of PLL3 were tested by hemagglutination assay, glycan array, isothermal titration calorimetry, and surface plasmon resonance, and its structure was determined by X-ray crystallography. Obtained data revealed that PLL3 binds similar carbohydrates to those that the other PLL family members bind, with some differences in the binding properties. PLL3 exhibited the highest affinity toward l-fucose and its derivatives but was also able to interact with O-methylated glycans and other ligands. Unlike the other members of this family, PLL3 was discovered to be a monomer, which might correspond to a weaker avidity effect compared to homologous lectins. Based on the similarity to the related lectins and their proposed biological function, PLL3 might accompany them during the interaction of P. laumondii with both the nematode partner and the insect host.

Fucosylated inhibitors of recently identified bangle lectin from Photorhabdus asymbiotica.

A recently described bangle lectin (PHL) from the bacterium Photorhabdus asymbiotica was identified as a mainly fucose-binding protein that could play an important role in the host-pathogen interaction and in the modulation of host immune response. Structural studies showed that PHL is a homo-dimer that contains up to seven L-fucose-specific binding sites per monomer. For these reasons, potential ligands of the PHL lectin: α-L-fucopyranosyl-containing mono-, di-, tetra-, hexa- and dodecavalent ligands were tested. Two types of polyvalent structures were investigated - calix[4]arenes and dendrimers. The shared feature of all these structures was a C-glycosidic bond instead of the more common but physiologically unstable O-glycosidic bond. The inhibition potential of the tested structures was assessed using different techniques - hemagglutination, surface plasmon resonance, isothermal titration calorimetry, and cell cross-linking. All the ligands proved to be better than free L-fucose. The most active hexavalent dendrimer exhibited affinity three orders of magnitude higher than that of standard L-fucose. To determine the binding mode of some ligands, crystal complex PHL/fucosides 2 - 4 were prepared and studied using X-ray crystallography. The electron density in complexes proved the presence of the compounds in 6 out of 7 fucose-binding sites.

Selectivity of original C-hexopyranosyl calix[4]arene conjugates towards lectins of different origin.

As a part of ongoing activities towards the design of ligands against pathogenic lectins, a synthesis of original α-C-galacto/α-C-manno/α-C-fucopyranosyl glycomimetics based on a calix[4]arene scaffold and their binding evaluation is described. The interactions of the glycomimetics with seven lectins of various origins were carried out using agglutination inhibition assays. The 1,3-alternate tetra-C-fucosylated ligand and its derivative having a tertBu group at the upper rim of the calix[4]arene scaffold were the most potent towards the AAL lectin family (RSL, AFL, AAL, AOL) and BC2L-C. As AFL and RSL originate from important human (Aspergillus fumigatus) and plant (Ralstonia solanacearum) pathogens, the inhibition potency of both leading structures was assessed by surface plasmon resonance. With AFL, both structures exhibited an approximately three orders of magnitude increase in affinity compared to the reference l-fucose. The role of tertBu groups as "aglycon-assisted" events was illustrated by NMR. Furthermore, both compounds showed significantly increased ability to inhibit BC2L-C (from human pathogen Burkholderia cenocepacia) cell agglutination and were able to cross-link whole B. cenocepacia cells. Although the ligands failed to significantly inhibit the agglutination activity of LecA and LecB from Pseudomonas aeruginosa, tetra-C-galactosylated calix[4]arene with tertBu groups at the upper rim of the 1,3-alternate conformation inhibited P. aeruginosa biofilm formation efficiently. This systematic and comprehensive study highlights the fact that hydrolytically stable polyvalent C-glycomimetics should be regarded as potent and selective ligands capable of acting as antiadhesive agents.

Polyvalent C-glycomimetics based on l-fucose or d-mannose as potent DC-SIGN antagonists.

The C-type lectin DC-SIGN expressed on immature dendritic cells is a promising target for antiviral drug development. Previously, we have demonstrated that mono- and divalent C-glycosides based on d-manno and l-fuco configurations are promising DC-SIGN ligands. Here, we described the convergent synthesis of C-glycoside dendrimers decorated with 4, 6, 9, and 12 α-l-fucopyranosyl units and with 9 and 12 α-d-mannopyranosyl units. Their affinity against DC-SIGN was assessed by surface plasmon resonance (SPR) assays. For comparison, parent O-glycosidic dendrimers were synthesized and tested, as well. A clear increase of both affinity and multivalency effect was observed for C-glycomimetics of both types (mannose and fucose). However, when dodecavalent C-glycosidic dendrimers were compared, there was no difference in affinity regarding the sugar unit (l-fuco, IC50 17 µM; d-manno, IC50 12 µM). For the rest of glycodendrimers with l-fucose or d-mannose attached by the O- or C-glycosidic linkage, C-glycosidic dendrimers were significantly more active. These results show that in addition to the expected physiological stability, the biological activity of C-glycoside mimetics is higher in comparison to the corresponding O-glycosides and therefore these glycomimetic multivalent systems represent potentially promising candidates for targeting DC-SIGN.

Nonhydrolyzable C-disaccharides, a new class of DC-SIGN ligands.

The discovery of effective ligands for DC-SIGN receptor is one of the most challenging concepts of antiviral drug design due to the importance of this C-type lectin in infection processes. DC-SIGN recognizes mannosylated and fucosylated oligosaccharides but glycosidic linkages are accessible to both chemical and enzymatic degradations. To avoid this problem, the synthesis of stable glycoside mimetics has attracted increasing attention. In this work we establish for the first time mono- and divalent C-glycosides based on d-manno and l-fuco configurations as prospective DC-SIGN ligands. In particular, the l-fucose glycomimetics were more active than the respective d-mannose ones. The highest affinity was assessed for simple 1,4-bis(α-l-fucopyranosyl)butane (SPR: IC50 0.43 mM) that displayed about twice higher activity than natural ligand Lex. Our results make C-glycosides attractive candidates for multivalent presentations.