Structural Basis of Carbohydrate Recognition by the Lectin LecB from Pseudomonas aeruginosa

doi:10.1016/S0022-2836(03)00754-X

Journal of Molecular Biology

Volume 331, Issue 4, 22 August 2003, Pages 861-870

https://doi.org/10.1016/S0022-2836(03)00754-X Get rights and content

Abstract

The crystal structure of Pseudomonas aeruginosa fucose-specific lectin LecB was determined in its metal-bound and metal-free state as well as in complex with fucose, mannose and fructopyranose. All three monosaccharides bind isosterically via direct interactions with two calcium ions as well as direct hydrogen bonds with several side-chains. The higher affinity for fucose is explained by the details of the binding site around C6 and O1 of fucose. In the mannose and fructose complexes, a carboxylate oxygen atom and one or two hydroxyl groups are partly shielded from solvent upon sugar binding, preventing them from completely fulfilling their hydrogen bonding potential. In the fucose complex, no such defects are observed. Instead, C6 makes favourable interactions with a small hydrophobic patch. Upon demetallization, the C terminus as well as the otherwise rigid metal-binding loop become more mobile and adopt multiple conformations.

Introduction

Lectins represent a specific class of carbohydrate-binding proteins different from enzymes or antibodies.¹ They are found in a wide range of organisms, including viruses, bacteria, plants and animals, and belong to one of several different families, many of which have been characterised structurally.2., 3., 4., 5. Bacterial lectins are believed to play an important role in infection, such as specific recognition of or attachment to target cells, as demonstrated for a mannose-specific lectin from Escherichia coli, which mediates the specific adhesion between the bacteria and many different human epithelal cell types.⁶ Furthermore, lectins may have a significant biotechnological and medical potential, as exemplified by the galactoside-specific mistletoe lectin, which is used on a large scale to support anti-cancer therapy.⁷ Structural data on bacterial lectins are limited. Only two pilus-associated adhesins from E. coli, FimH and PapG, have been characterised structurally.8., 9., 10.

Pseudomonas aeruginosa, an important opportunistic pathogen associated with chronic airway infections, synthesises two lectins, LecA and LecB (formerly named PA-IL and PA-IIL, respectively).¹¹ These lectins play an important role in human infections, since it was demonstrated that a P. aeruginosa-induced otitis externa diffusa¹² as well as several respiratory tract infections¹³ could be cured simply by treatment of the patients with a solution containing LecA and LecB-specific sugars. The sugar solutions presumably prevented the lectin-mediated bacterial adhesion to the corresponding host cells. The expression of the lectin genes in P. aeruginosa is co-ordinately regulated with certain other virulence factors and controlled via quorum sensing and by the alternative sigma factor RpoS.¹⁴

The galactophilic LecA has been characterised in great detail over the last 30 years. It consists of four 12.75 kDa subunits15., 16. and has been shown to cause cytotoxic effects on respiratory epithelial cells.¹⁷

LecB is an 11.73 kDa protein which exhibits a high specificity for (l)-fucose and its derivatives.18., 19. It decreases in vitro the ciliary beat frequency of the airway epithelium, hence inhibiting an important defence mechanism of the human lung.20., 21.

Both P. aeruginosa lectins were shown to be located mainly in the cytoplasm of planktonic cells.²² These findings make it difficult to explain the lectin-mediated cytotoxic and adhesive properties and have prompted us to investigate their cellular location in sessile P. aeruginosa cells. Our results showed that LecB is abundantly present in the bacterial outer membrane and, additionally, a LecB-deficient strain is significantly impaired in biofilm formation (D.T. et al., unpublished results). Since the formation of P. aeruginosa biofilms on tissues of infected patients as well as on medical devices was recently shown to be responsible for the inherent resistance of the bacteria to antibiotics and common antimicriobial agents,²³ we concluded that the knowledge of the LecB crystal structure and, in particular, of its carbohydrate-binding site may provide valuable information to develop potent anti-Pseudomonas therapeutics.

Recently, the structure of LecB was determined in complex with fucose.²⁴ Here, we present the refined crystal structures of the carbohydrate-free form of LecB, its complexes with mannose and fructose, and of calcium-depleted LecB.

Section snippets

Overall structure of LecB

We determined the crystal structure of LecB in its carbohydrate-free form, as a complex with mannose, mannotriose, fucose and fructose, and in its inactive calcium-free form. All structures are refined at high resolution (ranging from 2.0 Å to 1.2 Å) and with low R-factors (Table 1). The overall fold of LecB is a β-sandwich consisting of two antiparallel β-sheets of four and five strands, respectively (Figure 1(a)). This fold has been described in detail by other authors²⁴ and is topologically

Conclusion

Recently, Mitchell and co-workers²⁴ presented the crystal structure of LecB in complex with fucose, laying the foundations for developing anti-Pseudomonas therapeutics based on oligosaccharides. Our current study builds on this work by presenting the structures of LecB with different monosaccharide ligands, in its ligand-free state and in its inactive, calcium-free state. The two calcium ions not only directly interact with the bound monosaccharides, but also stabilize the conformation of loop

Bacterial strains and plasmids

The strains and plasmids used in this study are listed in Table 3. E. coli DH5α was used for cloning experiments and E. coli BL21(DE3) as a heterologous expression host for plasmid-encoded LecB.

Cloning and overexpression of LecB

A 345 bp DNA fragment was amplified by PCR using P. aeruginoa PAO1 DNA as the template with oligonucleotide primers LIINdeI (AAA ACA TAT GGC AAC ACA AGG AGT GTT CAC) and LIIBamHI (AAA AGG ATC CCT AGC CGA GCG GCC AGT TGA TC), introducing a BamHI or NdeI site, respectively. This fragment was digested with Nde

Acknowledgements

This work was supported by the Vlaams Interuniversitair Instituut voor Biotechnologie (VIB). R. L. is a postdoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO). K.-E. J. thanks the Mucoviscidose e.V. for financial support. The authors acknowledge the use of the EMBL beamlines at DESY (Hamburg, Germany and ESRF (Grenoble, France).

References (51)

R. Loris
Principles of structures of animal and plant lectins
Biochim. Biophys. Acta
(2002)
J. Bouckaert et al.
Novel structures of plant lectins and their complexes with carbohydrates
Curr. Opin. Struct. Biol.
(1999)
J.M. Rini et al.
New animal lectin structures
Curr. Opin. Struct. Biol.
(1999)
K.W. Dodson et al.
Structural basis of the pyelonephritic E. coli adhesin to its human kidney receptor
Cell
(2001)
N. Gilboa-Garber
Pseudomonas aeruginosa lectins
Methods Enzymol.
(1982)
N. Gilboa-Garber
Purification and properties of hemagglutinin from Pseudomonas aerginosa and its reaction with human blood cells
Biochim. Biophys. Acta
(1972)
D. Avichezer et al.
Analysis of the amino acid sequence of the Pseudomonas aeruginosa galactophilic PA-I lectin
J. Biol. Chem.
(1992)
N.C. Garber et al.
Specifity of the fucose-binding lectin of Pseudomonas aeruginosa
FEMS Microbiol. Letters
(1987)
J.W. Costerton
Cystic fibrosis pathogenesis and the role of biofilms in persistent infection
Trends Mircrobiol.
(2001)
C. Mattos
Protein–water interactions in a dynamic world
Trends Biochem. Sci.
(2002)

J.M. Rini et al.

X-ray crystal structure of a pea lectin-trimannoside complex at 2.6 Å resolution

J. Biol. Chem.

(1993)

R. Loris et al.

Conserved waters in legume lectin crystal structures. The importance of bound water for the sequence–structure relationship within the legume lectin family

J. Biol. Chem.

(1994)

R.T. Lee et al.

Ligand-binding characteristics of rat serum-type mannose-binding protein (MBP-A). Homology of binding site architecture with mammalian and chicken hepatic lectins

J. Biol. Chem.

(1991)

K.K.-S. Ng et al.

Orientation of bound ligands in mannose-binding proteins. Implications for multivalent lattice recognition

J. Biol. Chem.

(2002)

J. Bouckaert et al.

The structural features of concanavalin A governing non-proline peptide isomerization

J. Biol. Chem.

(2000)

J. Lescar et al.

Isolectins I-A and I-B of Griffonia (Bandeiraea) simplicifolia. Crystal structure of metal-free GS I-B(4) and molecular basis for metal binding and monosaccharide specificity

J. Biol. Chem.

(2002)

Z. Otwinowski et al.

Processing of X-ray diffraction data collected in oscillation mode

Methods Enzymol.

(1997)

E.A. Merritt et al.

Raster3D: photorealistic molecular graphics

Methods Enzymol.

(1997)

D. Hanahan

Studies on transformation of Escherichia coli with plasmids

J. Mol. Biol.

(1983)

F.W. Studier et al.

Use of bacteriophage T7 RNA polymerase to direct selective high level expression of cloned genes

J. Mol. Biol.

(1986)

S.H. Barondes

Bifunctional properties of lectins: lectins redefined

Trends Biochem. Sci.

(1988)

W.I. Weiss

Cell-surface carbohydrate recognition by animal and viral lectins

Curr. Opin. Struct. Biol.

(1997)

E.H. Beachey

Bacterial adherence: adhesin–receptor interactions mediating the attachment of bacteria to mucosal surface

J. Infect. Dis.

(1981)

J. Beuth et al.

Mistellektin-1: neue therapeutische Perspektiven in der Onkologie

Onkologie

(1995)

D. Choudhury et al.

X-ray structure of the FimC–FimH chaperone–adhesin complex from uropathogenic Escherichia coli

Science

(1999)

Cited by (108)

Surface chemistry dependent toxicity of inorganic nanostructure glycoconjugates on bacterial cells and cancer cell lines
2023, Journal of Drug Delivery Science and Technology
Citation Excerpt :
Under the “advanced search” button, “species” was selected as the criterion. P. aeruginosa: Mannose, fucose, and fructopyranose bind to LecB [25]; l-Galactose and d-arabinose bind to PA-IIL [26]; cinnamide derivatives can bind to LecB [27]; d-galactose can bind to PA-IIL [28]. E. coli: Phenyl ring possessing galactose can bind to heat-labile enterotoxin [29]; biphenyl-containing mannose can bind to FimH lectin [30].
Surface functionalized nanostructures have outstanding potential in biological applications owing to their target-specific design. In this study, we utilized laboratory synthesized carbohydrate-derivatives (i.e., galactose, mannose, lactose, and cellobiose derivatives) for aqueous one-pot synthesis of gold (Au) and silver (Ag) nanostructure glycoconjugates (NSs), and iron metal-organic framework glycoconjugates (FeMOFs). This work aims to test whether differences in the surface chemistry of the inorganic nanostructures play roles in revealing their toxicities towards bacterial cells and cancerous cell lines. As of the first step, biological activity of AuNSs, AgNSs, and FeMOFs were tested against a variety of gram (−) and gram (+) bacterial strains, where AgNSs possessed moderate to high antibacterial activities against all the tested bacterial strains, while AuNSs and FeMOFs showed their bacterial toxicity mostly depending on the strain. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) determination studies were performed for the nanostructure glycoconjugates, for which μg/mL MBC values were obtained such as (Cellobiose p-aminobenzoic acid_AgNS) CBpAB_AgNS gave 50 μg/mL MBC value for P.aeruginosa and S.kentucy. The activity of selected sugar ligands and corresponding glycoconjugates were further tested on MDA-MB-231 breast cancer and A549 lung cancer cell lines, where selective anticancer activity was observed depending on the surface chemistry as well. Besides, d-penicillamine was introduced to galectin specific sugar ligand coated AuNS glycoconjugates, which showed very strong anticancer activities even at low doses. Overall, the importance of this work is that the surface chemistry of the inorganic nanostructures can be critical to reveal their toxicity towards bacterial cells and cancerous cell lines.
Molecular and Mechanistic Basis of Lectin-Glycan Interactions
2021, Comprehensive Glycoscience: Second Edition
Recognition of glycoconjugates by lectins is critical for many biological processes. Lectins interact with glycans through hydrogen bonding, metal ion coordination, hydrophobic association, van der Waals and ionic interactions. Water molecules in the vicinity of the binding sites and glycan conformation also play roles. Furthermore, the valence of the glycan epitopes and the number and geometry of the binding sites of lectins play important roles in their interactions. Natural glycoconjugates are often multivalent and possess multiple copies of the same glycan epitope. Their multivalent interactions with lectins, which are oligomeric proteins, often enhance the weak binding affinities of a single glycan epitope. Multivalent interactions can be either intramolecular or intermolecular. In the latter case, gradients of microaffinity constants are associated with multivalent carbohydrates binding to lectins. When the valence of either the glycoconjugate or lectin exceeds two, intermolecular multivalent binding can result in lectin-glycoconjugate cross-linking interactions that form either organized or disorganized clustered complexes. Such complexes are involved in signal transduction mechanisms on the surface of cells. Scaffolds, the non-glycan parts of glycoconjugates, do not participate in lectin binding yet they modulate lectin binding interactions. In addition, lectins rarely to bind to sulfated glycosaminoglycans (GAGs) and proteoglycans. The human lectin Galectin-3 has the rare ability to do both. The binding affinity of lectins for linear biopolymers, such as mucins and GAGs, is proportional to the length of the polymers. A model termed “bind-and-jump” presented in this chapter explains how lectins interact with mucins and GAGs.
Synthesis of a calix[4]arene derivative exposing multiple units of fucose and preliminary investigation as a potential broad-spectrum antibiofilm agent
2019, Carbohydrate Research
Calix[4]arene derivative (1), bearing four α-l-C-fucosyl units linked via a flexible spacer, and a monomeric analogous (2) bearing a single moiety of fucose, were synthesized. Compounds 1 and 2 were assayed for antibiofilm activity against Pseudomonas aeruginosa (Gram–) and Staphylococcus epidermidis (Gram+). The macrocyclic compound 1 showed very high percentage of biofilm inhibition against two different bacterial strains while compound 2, which does not possess a macrocyclic structure, showed only moderate biofilm inhibition against P. aeruginosa and no biofilm inhibition against S. epidermidis. The fucose multivalent derivative could be a new broad-spectrum antibiofilm agent.
Glycoconjugate probes containing a core-fucosylated N-glycan trisaccharide for fucose lectin identification and purification
2017, Carbohydrate Research
Citation Excerpt :
Although scientists have recognized the importance of fucosylated glycoproteins in disease diagnosis and prognosis, the further functional studies on fucose involved biological events are always hampered by difficulty in identification and purification of in vivo fucose-binding proteins. So far, some fucose lectins derived from plants or bacteria were discovered, including AAL (Aleuria aurantia lectin) [23], LCA (Lens culinaris agglutinin-A) [24], AOL (Aspergillus oryzae L-fucose-specific lectin) [25], LecB from Pseudomonas aeruginosa [26], etc. Meanwhile, it is seldom reported on fucose-specific binding proteins or lectins isolated from mammalian animals.
Glyco-PAMAM dendrimers and glyco-agarose beads bearing a core-fucosylated N-glycan trisaccharide GlcNAcβ1,4(Fucα1,6)GlcNAc or a non-fucose disaccharide GlcNAcβ1,4GlcNAc were successfully synthesized and characterized by monosaccharide analysis with HPAEC-PAD technique. These glycoconjugates as fucose lectin probes were applied in fucose-specific lectin detection and purification. The model fucose lectin AAL indicated binding activity with the FITC-labeled PAMAM carrying core-fucose trisaccharide. An affinity chromatography column stuffed with the agarose beads carrying core-fucosylated trisaccharide exhibited a good specificity in purification of AAL than non-fucose disaccharide agarose beads. These novel glycoconjugates bearing the precise and simplified core-fucose N-glycan structure provided a potential application for core-fucose-specific lectin discovery.
Glycomimetics for the inhibition and modulation of lectins
2023, Chemical Society Reviews
Aptamers as Novel Binding Molecules on an Antimicrobial Peptide-Armored Composite Hydrogel Wound Dressing for Specific Removal and Efficient Eradication of Pseudomonas aeruginosa
2023, International Journal of Molecular Sciences

View all citing articles on Scopus

View full text

Journal of Molecular Biology

Structural Basis of Carbohydrate Recognition by the Lectin LecB from Pseudomonas aeruginosa

Abstract

Introduction

Section snippets

Overall structure of LecB

Conclusion

Bacterial strains and plasmids

Cloning and overexpression of LecB

Acknowledgements

Biochim. Biophys. Acta

Curr. Opin. Struct. Biol.

Curr. Opin. Struct. Biol.

Cell

Methods Enzymol.

Biochim. Biophys. Acta

J. Biol. Chem.

FEMS Microbiol. Letters

Trends Mircrobiol.

Trends Biochem. Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

Methods Enzymol.

J. Mol. Biol.

J. Mol. Biol.

Bifunctional properties of lectins: lectins redefined

Trends Biochem. Sci.

Cell-surface carbohydrate recognition by animal and viral lectins

Curr. Opin. Struct. Biol.

Bacterial adherence: adhesin–receptor interactions mediating the attachment of bacteria to mucosal surface

J. Infect. Dis.

Mistellektin-1: neue therapeutische Perspektiven in der Onkologie

Onkologie

X-ray structure of the FimC–FimH chaperone–adhesin complex from uropathogenic Escherichia coli

Science