Catherine Gautier

Structural basis for oligosaccharide-mediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients

Pseudomonas aeruginosa galactose- and fucose-binding lectins (PA-IL and PA-IIL) contribute to the virulence of this pathogenic bacterium, which is a major cause of morbidity and mortality in cystic fibrosis patients. The crystal structure of PA-IIL in complex with fucose reveals a tetrameric structure. Each monomer displays a nine-stranded, antiparallel b-sandwich arrangement and contains two close calcium cations that mediate the binding of fucose in a recognition mode unique among carbohydrate–protein interactions. Experimental binding studies, together with theoretical docking of fucose-containing oligosaccharides, are consistent with the assumption that antigens of the Lewis a (Lea) series may be the preferred ligands of this lectin. Precise knowledge of the lectin-binding site should allow a better design of new antibacterial-adhesion prophylactics.

Structural Basis of Calcium and Galactose Recognition by the Lectin Pa-Il of Pseudomonas Aeruginosa

The structure of the tetrameric Pseudomonas aeruginosa lectin I (PA‐IL) in complex with galactose and calcium was determined at 1.6 Å resolution, and the native protein was solved at 2.4 Å resolution. Each monomer adopts a β‐sandwich fold with ligand binding site at the apex. All galactose hydroxyl groups, except O1, are involved in a hydrogen bond network with the protein and O3 and O4 also participate in the co‐ordination of the calcium ion. The stereochemistry of calcium galactose binding is reminiscent of that observed in some animal C‐type lectins. The structure of the complex provides a framework for future design of anti‐bacterial compounds.

Structural Basis for the Interaction between Human Milk Oligosaccharides and the Bacterial Lectin Pa-Iil of Pseudomonas Aeruginosa.

One of the mechanisms contributing to the protection by breast-feeding of the newborn against enteric diseases is related to the ability of human milk oligosaccharides to prevent the attachment of pathogenic bacteria to the duodenual epithelium. Indeed, a variety of fucosylated oligosaccharides, specific to human milk, form part of the innate immune system. In the present study, we demonstrate the specific blocking of PA-IIL, a fucose-binding lectin of the human pathogen Pseudomonas aeruginosa, by milk oligosaccharides. Two fucosylated epitopes, Lewis a and 3-fucosyl-lactose (Lewis x glucose analogue) bind to the lectin with dissociation constants of 2.2x10(-7) M and 3.6x10(-7) M respectively. Thermodynamic studies indicate that these interactions are dominated by enthalpy. The entropy contribution is slightly favourable when binding to fucose and to the highest-affinity ligand, Lewis a. The high-resolution X-ray structures of two complexes of PA-IIL with milk oligosaccharides allow the precise determination of the conformation of a trisaccharide and a pentasaccharide. The different types of interaction between the oligosaccharides and the protein involve not only hydrogen bonding, but also calcium- and water-bridged contacts, allowing a rationalization of the thermodynamic data. This study provides important structural information about compounds that could be of general application in new therapeutic strategies against bacterial infections.

An Unusual Carbohydrate Binding Site Revealed by the Structures of Two Maackia amurensis Lectins Complexed with Sialic Acid-containing Oligosaccharides

Seeds from the legume tree Maackia amurensis contain two lectins that can agglutinate different blood cell types. Their specificity toward sialylated oligosaccharides is unique among legume lectins; the leukoagglutinin preferentially binds to sialyllactosamine (αNeuAc(2–3)βGal(1–4)βGlcNAc), whereas the hemagglutinin displays higher affinity for a disialylated tetrasaccharide (αNeuAc(2–3)βGal(1–3)[αNeuAc(2–6)]αGalNAc). The three-dimensional structure of the complex between M. amurensis leukoagglutinin and sialyllactose has been determined at 2.75-Å resolution using x-ray crystallography. The carbohydrate binding site consists of a deep cleft that accommodates the three carbohydrate residues of the sialyllactose. The central galactose sits in the primary binding site in an orientation that has not been observed previously in other legume lectins. The carboxyl group of sialic acid establishes a salt bridge with a lysine side chain. The glucose residue is very efficiently docked between two tyrosine aromatic rings. The complex between M. amurensishemagglutinin and a disialylated tetrasaccharide could be modeled from the leukoagglutinin/sialyllactose crystal structure. The substitution of one tyrosine by an alanine residue is responsible for the difference in fine specificity between the two isolectins. Comparison with other legume lectins indicates that oligosaccharide specificity within this family is achieved by the recycling of structural loops in different combinations.

High affinity fucose binding of Pseudomonas aeruginosa lectin PA‐IIL: 1.0 Å resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches

PA‐IIL is a fucose‐binding lectin from Pseudomonas aeruginosa that is closely related to the virulence factors of the bacterium. Previous structural studies have revealed a new carbohydrate‐binding mode with direct involvement of two calcium ions (Mitchell E, Houles C, Sudakevitz D, Wimmerova M, Gautier C, Peréz S, Wu AM, Gilboa‐Garber N, Imberty A. Structural basis for selective recognition of oligosaccharides from cystic fibrosis patients by the lectin PA‐IIL of Pseudomonas aeruginosa. Nat Struct Biol 2002;9:918–921). A combination of thermodynamic, structural, and computational methods has been used to study the basis of the high affinity for the monosaccharide ligand. A titration microcalorimetry study indicated that the high affinity is enthalpy driven. The crystal structure of the tetrameric PA‐IIL in complex with fucose and calcium was refined to 1.0 Å resolution and, in combination with modeling, allowed a proposal to be made for the hydrogen‐bond network in the binding site. Calculations of partial charges using ab initio computational chemistry methods indicated that extensive delocalization of charges between the calcium ions, the side chains of the protein‐binding site and the carbohydrate ligand is responsible for the high enthalpy of binding and therefore for the unusually high affinity observed for this unique mode of carbohydrate recognition. Proteins 2005.

Binding of different monosaccharides by lectin PA-IIL from Pseudomonas aeruginosa : Thermodynamics data correlated with X-ray structures

The lectin from Pseudomonas aeruginosa (PA‐IIL) is involved in host recognition and biofilm formation. Lectin not only displays an unusually high affinity for fucose but also binds to l‐fucose, l‐galactose and d‐arabinose that differ only by the group at position 5 of the sugar ring. Isothermal calorimetry experiments provided precise determination of affinity for the three methyl‐glycosides and revealed a large enthalpy contribution. The crystal structures of the complexes of PA‐IIL with l‐galactose and Met‐β‐d‐arabinoside have been determined and compared with the PA‐IIL/fucose complex described previously. A combination of the structures and thermodynamics provided clues for the role of the hydrophobic group in affinity.

High affinity interaction between a bivalve C-type lectin and a biantennary complex-type N-glycan revealed by crystallography and microcalorimetry.

Codakine is an abundant 14-kDa mannose-binding C-type lectin isolated from the gills of the sea bivalve Codakia orbicularis. Binding studies using inhibition of hemagglutination indicated specificity for mannose and fucose monosaccharides. Further experiments using a glycan array demonstrated, however, a very fine specificity for N-linked biantennary complex-type glycans. An unusually high affinity was measured by titration microcalorimetry performed with a biantennary Asn-linked nonasaccharide. The crystal structure of the native lectin at 1.3Å resolution revealed a new type of disulfide-bridged homodimer. Each monomer displays three intramolecular disulfide bridges and contains only one calcium ion located in the canonical binding site that is occupied by a glycerol molecule. The structure of the complex between Asn-linked nonasaccharide and codakine has been solved at 1.7Å resolution. All residues could be located in the electron density map, except for the capping β1–4-linked galactosides. The α1–6-linked mannose binds to calcium by coordinating the O3 and O4 hydroxyl groups. The GlcNAc moiety of the α1,6 arm engages in several hydrogen bonds with the protein, whereas the GlcNAc on the other antenna is stacked against Trp108, forming an extended binding site. This is the first structural report for a bivalve lectin.

Stereochemical analysis of d-glucopyranosyl-sulfoxides via a combined NMR, molecular modeling and X-ray crystallographic approach

Abstract ( S )-α-Methoxyphenylacetic acid (MPAA) was used as an NMR shift reagent in combination with molecular modeling to predict the absolute configuration of a representative epimeric pair of glucopyranosyl sulfoxides. The correctness of this assignment was confirmed by X-ray crystallographic examination of one of the epimers, 3a1 . The crystal structure of ethyl 2,3-di- O -acetyl-4,6- O -benzylidene-1-thio-β- d -glucopyranoside S -oxide monohydrate 3a1 was solved by direct methods and was shown to bear the ( R )-configuration at the sulfinyl center in accordance with our prediction. Furthermore, the conformation of 3a1 in the solid state was found to be remarkably similar to that predicted by molecular mechanics calculations.

Characterization of the binding of α-L-Fuc (1->2)-β-D-Gal (1->), a xyloglucan signal, in blackberry protoplasts.

Abstract Previous work showed that the fucose→galactose moiety of the xyloglucan nonasaccharide XXFG is responsable for its biological activity. We used this side chain of XXFG (α- l -Fuc (1→2)-β- d -Gal (1→)) in ligand-binding experiments to demonstrate its role as a signal molecule in plant cells. Proteins solubilized from plasma membrane enriched fractions isolated from Rubus fruticosus protoplasts were tested for their ability to bind the side chain of XXFG, using a digoxigenin- or biotin-conjugated neoglycoprotein specific for 2′-fucosyl-lactose in blots and k-ELISA tests. A putative receptor for the signaling sugar was identified, and the ligand specificity is reported. The role of structural elements important for biological activities was investigated using compounds structurally related to xyloglucan, and a variety of phytohormones such as 2,4-D.

Immobilized sugars as abiotic inducers of β-d-glycanohydrolases in plant cells

Abstract Silica beads coated with various sugars were used to induce β- d -glycanohydrolases in Rubus-cultured cells. Laminarase, xylanase and chitosanase activities were strongly increased after the incubation of cells for 1 h with the immobilized sugars. The chemical structure and the dose of immobilized sugars affect the nature and the extent of the induced activities. Optimal induction of enzymes required only about 100 nmol of sugar per g cells and occurred within 1 h cell-treatment. The activation of β- d -glycanohydrolases could be modulated in presence of Ca2+ or K+ regulators. The findings suggest that the immobilized sugars mimic some effects of biotic elicitoes derived from pathogen or plant cell-walls.