A structural biology approach to understand human lymphatic filarial infection

Abstract

The presence of aspartic protease inhibitor in filarial parasite Brugia malayi (Bm-Aspin) makes it interesting to study because of the fact that the filarial parasite never encounters the host digestive system. Here, the aspartic protease inhibition kinetics of Bm-Aspin and its NMR structural characteristics have been investigated. The overall aim of this study is to explain the inhibition and binding properties of Bm-Aspin from its structural point of view. UV-spectroscopy and multi-dimensional NMR are the experiments that have been performed to understand the kinetic and structural properties of Bm-Aspin respectively. The human aspartic proteases that are considered for this study are pepsin, renin, cathepsin-E and cathepsin-D. The results of this analysis performed with the specific substrate [Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu (4-pyridylmethyl) ester] against aspartic proteases suggest that Bm-Aspin inhibits the activities of all four human aspartic proteases. The kinetics studies indicate that Bm-Aspin follows a competitive mode of inhibition for pepsin and cathepsin-E, non-competitive for renin and mixed mode for cathepsin-D. The triple resonance NMR experiments on Bm-Aspin suggested the feasibility of carrying out NMR studies to obtain its solution structure. The NMR titration studies on the interactions of Bm-Aspin with the proteases indicate that it undergoes fast-exchange phenomena among themselves. In addition to this, the chemical shift perturbations for some of the residues of Bm-Aspin observed from (15)N-HSQC spectra upon the addition of saturated amounts of aspartic proteases suggest the binding between Bm-Aspin and human aspartic proteases. They also provide information on the variations in the intensities and mode of binding between the proteases duly corroborating with the results from the protease inhibition assay method.

Figure 1. Human aspartic protease inhibition activity.

(A) The inhibition activity of Bm-Aspin against human aspartic proteases using UV-spectroscopy; SU: Substrate, P: Pepsin, RE: Renin, CE: Cathepsin-E, CD: Cathepsin-D, PS: Pepstatin, Bm: Bm-Aspin. The values represent the mean of six independent experiments ± SD. (B). The activity of pepsin and inhibition of Bm-Aspin in the presence of 100 mM SDS using Casein agar plate: (i) at pH 5.6: Reaction Buffer (100 mM sodium acetate, 10 mM CaCl₂) (well 1), Water (well 2), 5 µg of pepsin (well 3), Equimolar Bm-Aspin and pepsin preincubated for 10 minutes at 37°C (well 4), 5 µg of pepsin with 5 mM pepstatin (well 5), 5 µg of pepsin preincubated with 100 mM SDS for 10 minutes at 37°C (well 6), Bm-Aspin with SDS treated pepsin (well 7), pepstatin with SDS treated pepsin (well 8) and (ii) at pH 7.0: Reaction Buffer (25 mM phosphate buffer, 10 mM CaCl₂) (well 1), Well 2 to Well 8 contain the same as before. The zone of digestion by pepsin is indicated by a bold dash. (C). The activity of pepsin and inhibition of Bm-Aspin in the presence of 100 mM SDS with pH 5.6 and 7.0 using UV-spectroscopy: SU: substrate, P: pepsin, PS: pepstatin, Bm: Bm-Aspin, SD: SDS. The plots in green and red represent the activity of pepsin at pH 5.6 and 7.0 respectively. The values represent the mean of three independent experiments ± SD.

Figure 2. Kinetics of aspartic protease inhibition by Bm-Aspin.

Lineweaver-Burk Plots showing the variation (1/V with that of 1/S) of competitive inhibition of pepsin (A) and cathepsin-E (C), non-competitive for renin (B) and mixed inhibition for cathepsin-D (D) respectively. Assays were carried out in triplicates, with the fixed quantity of proteases (5 mM) and varying concentrations of Bm-Aspin (0 mM, 1 mM, 2.5 mM and 5 mM) The inhibition constants were determined using Graphpad Prism 2.0 (San Diego, CA).

Figure 3. NMR screening on Bm-Aspin with different detergents.

¹⁵N HSQC spectra of Bm-Aspin at pH 7.0 with the addition of: (i) No detergent (ii) 0.5 M Urea and 1% glycerol (iii) 1% n-octyl-β-D-glucoside (OG), (iv) 100 mM n-Dodecyl β-D-Maltopyranoside (DDM) (v) 1% triton X-100, and (vi) 100 mM SDS.

Figure 4. Bm-Aspin ¹⁵N HSQC spectra at varying concentrations of SDS.

Comparison of Bm-Aspin ¹⁵N HSQC spectra in the presence of varying concentrations of SDS; (i) 50 mM SDS, (ii) 100 mM SDS, (iii) 150 mM SDS, and (iv) 200 mM SDS. The inset box indicates the well resolved glycine peaks for comparison to identify the optimum solvent conditions for a well behaved NMR spectrum.

Figure 5. Sequential residue connectivities using triple resonance NMR strip plot.

Plot showing the strips of four triple resonance NMR spectra of Bm-Aspin in the following order: HNCOCA, HNCA, CBCACONH and HNCACB. The sequential ¹³Cα connectivities for the residues' stretch T189-V194 are indicated by the continuous line drawn between the adjacent Cα.

Figure 6. NMR chemical shift perturbations in Bm-Aspin due to pepsin interactions.

Perturbations in the chemical shift position of the residues Y215 (a), I214 (b), and A213 (c) respectively in Bm-Aspin upon addition of increasing concentrations of pepsin. Ratios of Bm-Aspin to pepsin are: 1∶0 (red), 1∶0.1 (cyan), 1∶0.5 (green), and 1.1 (yellow).

Figure 7. Chemical shift perturbations upon addition of human aspartic proteases.

(A) NMR Chemical shift Perturbations in Bm-Aspin due to different protease interactions at their saturated conditions. Chemical shift perturbations of the residues Y215 (i), I214 (ii), and A213 (iii) respectively, upon the addition of human aspartic proteases at their saturation levels. Free Bm-Aspin (Magenta), Bm-Aspin+Cathepsin-D (Yellow), Bm-Aspin+Cathepsin-E (Green), Bm-Aspin+Renin (Red), Bm-Aspin+Pepsin (Grey). (B) Comparison of Bm-Aspin chemical shift perturbation upon addition of human aspartic proteases. The bar diagram indicating the radial shift (calculated for each of the affected residues, by combining both the chemical shifts of ¹H and ¹⁵N, using the equation: Radial shift displacement (Δδ) = {(H_f−H_b) ²+[(N_f−N_b)/6] ²} ^1/2. A scaling factor of 6 was used to normalize the differences in the ¹H and ¹⁵N spectral widths. H_f, H_b, N_f, and N_b are the chemical shifts of each residue's amide ¹H and ¹⁵N in the free (Bm-Aspin alone) and bound (Bm-Aspin+protease complex) states, respectively) in ppm, observed due to NMR chemical shift perturbations in Bm-Aspin with the addition of proteases, (Bm-Aspin+Pepsin in blue, Bm-Aspin+Renin in red, Bm-Aspin+Cathepsin-E in green, Bm-Aspin+Cathepsin-D in magenta), observed for the following 10 residues: G16, G22, G82, G169, G190, A192, A204, A213, I214, and Y215.