Figure 1.
A) Peptide sequence of Bc2L-C with N-terminal domain in black, linker in green and C-terminal domain in red. B) Sequence alignment of the C-terminal domain of BC2L-C with related lectins from Burkholderia cenocepacia, Pseudomonas aeruginosa, Ralstonia solanacearum and Chromobacterium violaceum. Grey shading indicates the conservation of amino acids (black for fully conserved). Amino acids involved in calcium binding are indicated by diamonds. Amino acids predicted to form hydrogen bonds with the carbohydrate ligand are indicated by dots.
Figure 2.
SPR sensorgrams of BC2L-C and C-ter domain on different monosaccharide-activated surfaces.
The whole lectin binds efficiently to CM5 chips covered with PAA-fucose and PAA-mannose (left panel), while the isolated C-terminal domain has a strong specificity for fucose (right panel). In both cases, control curves obtained with galactose-modified channels have been subtracted.
Figure 3.
Glycan array data obtained with the whole BCL2-C lectin and the two domains expressed separately.
Purified BC2L-C lectin samples were labeled with Alexa Fluor and tested on Glycan Array of the Consortium for Functional Glycomics. The BC2L-C-ct and BCLC-nt domains bind specifically to mannosylated and fucosylated oligosaccharides, respectively while the whole lectin binds to both.
Table 1.
Microcalorimetry titration data for the binding of monosaccharides and oligosaccharides to BC2L-C and BC2L-C-ct.
Figure 4.
Crystal structure of apoBC2L-C C-terminal domain.
A. Dimeric organisation of apo-BC2L-C-Ct, the protein is represented as ribbon. The sulphate ion close to one of the calcium and monosaccharide binding site is represented as sticks. B. Open conformation of the binding site in the absence of calcium and ligand. The sulphate ion interacts with Gln204, His177 and water molecules. C. Molecular modelling of BC2L-C-Ct in complex with calcium and α-methyl-mannoside with rearrangement of binding amino acids. D. Model of the complex with α-methyl-heptoside.
Table 2.
Data collection and refinement statistics of BC2L-C-ct crystal structure.
Figure 5.
Three-dimensional reconstruction of BC2L-C.
A. Refined ab-initio SAXS envelop. B. Isosurface representation of the EM reconstruction. C. Superposition of EM reconstruction with the SAXS envelope. D. Ten projections of the EM reconstruction (upper row) are shown for a visual comparison with the class averages (lower row).
Figure 6.
Models of the BC2L-C hexamer with N-terminal domains in blue and C-terminal domains in green.
A. Best manual fit of the different domains of BC2L-C hexamer in the ab-initio SAXS envelop. B. Two possible arrangements of trimers that could correspond to the manual fit. C. Orthogonal orientations of the mode I. Linkers have been schematized by dotted lines. Calcium and carbohydrate ligands (fucose for the N-terminus and mannose for C-terminus) are represented by spheres.
Figure 7.
Immunodetection of soluble lectins.
10 µg of purified lectins were separated on a 16% SDS-PAGE, and the gel stained with Coomassie Blue or transferred to nitrocellulose membranes that were reacted with anti-cepacia polyclonal rabbit antibodies. Reacting bands were detected by fluorescence with an Odyssey infrared imaging system (Li-cor Biosciences) using IRDye800CW affinity purified anti-rabbit IgG antibodies (Rockland, Pennsylvania). M, prestained molecular weight standards. The star indicates the presence of a weak bank for Bcl2L-A.
Figure 8.
Localisation of B. cenocepacia soluble lectins in bacterial cells.
A. BC2L-A, -B and –C are secreted into the growth medium. Analysis of concentrated culture supernatants and total cell lysates recovered from B. cenocepacia K56-2 containing the control plasmid pEL-1 (lanes 1 and 5), K56-2 pBC2L-A-FLAG (lanes 2 and 6), K56-2 pBC2L-B-FLAG (lanes 3 and 7) and K56-2 pBC2L-C-FLAG (lanes 4 and 8). B. BC2L-B and –C are released from the bacterial surface upon mannose treatment. Analysis of concentrated supernatants from cells treated with PBS or 50 mM d-mannose and total cell lysates recovered from B. cenocepacia K56-2 pBC2L-A-FLAG (lanes 1), K56-2 pBC2L-B-FLAG (lanes 2) and K56-2 pBC2L-C-FLAG (lanes 3). Western blots were performed using anti-FLAG and anti-RNAP alpha subunit antibodies (cell lysis control). A degradation product from BC2L-C can be detected in the total cell lysates. The arrow highlights the position of the RNAP alpha subunit in total cell extracts. Samples were boiled 10 min prior to loading on 18% SDS-PAGE gels.
Figure 9.
Activation of respiratory epithelial cells by BC2L-C and its separate domains.
Sub-confluent BEAS-2B cells cultured in 24-well plates were incubated in 300 µL medium with either BC2L-C, BC2L-C-nt or BC2L-C-ct at 0.3 µM. As negative and positive controls, cells were either not stimulated (NS), incubated with the suspension buffer of BC2L-C (buffer) or challenged with 10 ng/mL of TNF-α (TNF). After 15 h, supernatants were collected and IL-8 concentrations were measured by ELISA. Each histogram is the mean ± sem of 3 experiments performed in triplicate.
Figure 10.
Schematic representation of BC2L-C hexamer cross-linking host epithelial cells and bacteria surface.