Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines

Abstract

A protein isolated from the bark of Crataeva tapia (CrataBL) is both a Kunitz-type plant protease inhibitor and a lectin. We have determined the amino acid sequence and three-dimensional structure of CrataBL, as well as characterized its selected biochemical and biological properties. We found two different isoforms of CrataBL isolated from the original source, differing in positions 31 (Pro/Leu); 92 (Ser/Leu); 93 (Ile/Thr); 95 (Arg/Gly) and 97 (Leu/Ser). CrataBL showed relatively weak inhibitory activity against trypsin (Kiapp = 43 µM) and was more potent against Factor Xa (Kiapp = 8.6 µM), but was not active against a number of other proteases. We have confirmed that CrataBL contains two glycosylation sites and forms a dimer at high concentration. The high-resolution crystal structures of two different crystal forms of isoform II verified the β-trefoil fold of CrataBL and have shown the presence of dimers consisting of two almost identical molecules making extensive contacts (∼645 Å(2)). The structure differs from those of the most closely related proteins by the lack of the N-terminal β-hairpin. In experiments aimed at investigating the biological properties of CrataBL, we have shown that addition of 40 µM of the protein for 48 h caused maximum growth inhibition in MTT assay (47% of DU145 cells and 43% of PC3 cells). The apoptosis of DU145 and PC3 cell lines was confirmed by flow cytometry using Annexin V/FITC and propidium iodide staining. Treatment with CrataBL resulted in the release of mitochondrial cytochrome c and in the activation of caspase-3 in DU145 and PC3 cells.

Figure 1. Purification of 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).

Figure 2. Similarity of CrataBL to others proteins.

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).

Figure 3. Molecular mass of glycosylated CrataBL determined by MALDI-TOF/MS.

Figure 4. Similarity of CrataBL to protease inhibitors.

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.

Figure 5. Electron density at the C terminus of molecule A of crystal form I of CrataBL.

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 .

Figure 6. Overall structure of 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 .

Figure 7. A comparison of CrataBL with proteins sharing the β-trefoil fold.

(A) Superimposed regions of the N termini of CrataBL (green) and STI (magenta - PDB ID: 1AVW [44]). (B) A dimer of CrataBL (red) compared to the obligatory dimers of CNL (yellow - PDB ID: 3NBD [9]) and GalNAc/Gal-specific agglutinin from Sclerotinia sclerotiorum (green - PDB ID: 2X2S [50]).

Figure 8. Effects of CrataBL and SbTI on the viability of the DU145 (A, C) and PC3 (B, D) cell lines, respectively.

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%.

Figure 9. CrataBL induced apoptosis in prostate cancer cells.

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).

Figure 10. Scanning confocal microscopy of DU145 (A) and PC3 (B) cells (5×10⁴/well).

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.

Figure 11. Analysis of caspase-3 activation in prostate cancer cell lines.

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.

Figure 12. Semi-transparent space-filling representation of the surface of CrataBL molecule colored by residue charges (red negative, blue positive).

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 .