PMC

Arginine and lysine residues which create a contiguous, positively charged channel which may be utilized for binding of sulfated oligosaccharides are labeled. Figure prepared with PyMol .

(A) Superimposed regions of the N termini of CrataBL (green) and STI (magenta - PDB ID: 1AVW ). (B) A dimer of CrataBL (red) compared to the obligatory dimers of CNL (yellow - PDB ID: 3NBD ) and GalNAc/Gal-specific agglutinin from Sclerotinia sclerotiorum (green - PDB ID: 2X2S ).

The 2F_o-F_c map was contoured at 1.2 σ level. The shape of the density corresponding to the C-terminal carboxylate clearly indicates that Gly165, although found in the sequence, is not present in the isoform forming the crystal. Figure prepared with PyMol .

A comparison of the amino acid sequence of isoform I of CrataBL with the sequences of BvTI – trypsin inhibitor purified from Bauhinia variegata; BuXI – inhibitor of factor Xa from Bauhinia ungulata; EcTI – trypsin inhibitor purified from Enterolobium contortisiliquum trypsin inhibitor , . Cysteine residues are shown in gray and highly conserved amino acids are highlighted in black. Residue positions P1 and P1′, between which the proteolytic cleavage occurs, are denoted in boxes. Asterisks indicate glycosylation sites in CrataBL.

(A) Stereo view of the three-dimensional structure of a CrataBL monomer (molecule A of crystal form I). Three motifs defining the β-trefoil fold are colored in blue, green, and red. N- and C-termini and the putative reactive loop are labeled. Two glycosylated residues and attached carbohydrates are shown in sticks. The carbohydrate attached to the reactive loop is taken from molecule A in crystal form II. (B) A dimer of CrataBL observed in both crystal forms, with the two molecules colored red and blue, respectively. The reactive loops in both monomers are shown in gray. The secondary structure elements are marked on the molecule shown on the left. Figure prepared with PyMol .

A comparison of the amino acid sequence of isoform I of CrataBL with the sequences of structurally similar proteins, as determined with the program Dali . Cysteine residues are shown in black boxes and highly conserved amino acids are highlighted in gray. Residue positions P1 and P1′, between which the proteolytic cleavage occurs, are denoted in box. Asterisks indicate glycosylation sites in CrataBL. The sequence of isoform II differs in positions 31 (Pro/Leu); 92 (Ser/Leu); 93 (Ile/Thr); 95 (Arg/Gly) and 97 (Leu/Ser).

The cell lines were seeded in 6-well plates for 24 h, followed by washing three times and incubating with RPMI without FBS for cell cycle synchronization. Afterwards cells were treated with CrataBL (40 µM) or medium (control) for 24 h (A and B) and 48 h (C and D). The analysis was performed in FACSCalibur flow cytometer using annexin V-FITC and propidium iodide (PI) staining. The percentage of viable (An⁻PI⁻), apoptotic (An⁺PI⁻), secondary apoptotic (An⁺PI⁺) and necrotic (An⁻PI⁺) cells are represented. Data expressed as mean ± SD, obtained from experiments performed in triplicate. (*p<0.05; *** p<0.001 compared with control cells).

DU145 (A) and PC3 (B) (1×10⁵ cells) cell lines were seeded in 6-well plates, following the same protocol for apoptosis with annexin V/FITC and PI staining. Cells treated with CrataBL (40 µM), containing RPMI without FBS were incubated for 48 h at 37°C and 5% (v/v) CO₂. The cells were incubated with 10 µL of cleaved caspase 3 Alexa Fluor 488-conjugated antibody for 40 min and analyzed in FACSCalibur flow cytometer. As control, the cells were treated with medium only. The area in black represents the control and in white, cells treated with CrataBL.

(A) The fraction 30–60% was submitted to ion-exchange chromatography on CM cellulose equilibrated with 10 mM phosphate citrate buffer (PCB), pH 5.5. Two mL fractions were collected at the flow rate 0.3 mL/min. An arrow indicates elution with 10 mM phosphate citrate buffer, pH 5.5, containing 0.5 M NaCl; (B) Size exclusion chromatography on Superdex 75 equilibrated with 0.15 M NaCl at the flow rate 0.5 mL/min. (C) The protein fraction was eluted with a linear gradient (5–100%) of 90% acetonitrile in 0.1% TFA in Milli-Q water (solvent B) at the flow rate of 0.7 mL/min (t = 0.1 min, 5% B; t = 5 min, 5% B; t = 30 min, 40% B; t = 50 min, 50% B; t = 60 min, 100% B; t = 65–68 min, 0% B).

Cells were incubated in 96-well microplates in the concentration of 5×10³ cells/100 µL/well. After 24 h, CrataBL, in the final concentrations of 5, 10, 20 and 40 µM, was incubated at 37°C in an atmosphere of 5% (v/v) CO₂ for 24, 48 and 72 h. The viability was determined by MTT colorimetric method. Results represent the mean ± standard deviation of three experiments, each conducted in triplicate (*p<0.05; ** p<0.01; *** p<0.001 compared with control cells). In the absence of CrataBL, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction was considered as 100%.

The cells were seeded on 13-mm glass coverslips, placed in 24-well plates and incubated for 24 h at 37°C, in an atmosphere of 5% (v/v) CO₂. At the end of the incubation period, the cells were incubated with RPMI without FBS for 24 h. Subsequently the cells were treated with 40 µM of CrataBL (12 h) in RPMI without FBS at 37°C, in an atmosphere of 5% (v/v) CO₂. The cells were washed with PBS and incubated with Mitotracker Deep Red 633 in the dark. The cell lines were marked with a primary antibody mouse anti-cytochrome c, following the incubation with a secondary antibody mouse anti-IgG conjugated with Alexa Fluor 488 (green). The nuclei were marked with DAPI (blue) and the coverslips fixed to slides with fluoromount G. The images were acquired using the confocal scanning microscope Carl Zeiss LSM780. Bar = 10 µm.