Differences in Charge Distribution in Leishmania tarentolae Leishmanolysin Result in a Reduced Enzymatic Activity

Abstract

Leishmania tarentolae is a non-pathogenic trypanosomatid isolated from lizards widely used for heterologous protein expression and extensively studied to understand the pathogenic mechanisms of leishmaniasis. The repertoire of leishmanolysin genes was reported to be expanded in L. tarentolae genome, but no proteolytic activity was detected. Here, we analyzed L. tarentolae leishmanolysin proteins from the genome to the structural levels and evaluated the enzymatic activity of the wild-type and overexpressing mutants of leishmanolysin. A total of 61 leishmanolysin sequences were retrieved from the L. tarentolae genome. Five of them were selected for phylogenetic analysis, and for three of them, we built 3D models based on the crystallographic structure of L. major ortholog. Molecular dynamics simulations of these models disclosed a less negative electrostatic potential compared to the template. Subsequently, L. major LmjF.10.0460 and L. tarentolae LtaP10.0650 leishmanolysins were cloned in a pLEXSY expression system into L. tarentolae. Proteins from the wild-type and the overexpressing parasites were submitted to enzymatic analysis. Our results revealed that L. tarentolae leishmanolysins harbor a weak enzymatic activity about three times less abundant than L. major leishmanolysin. Our findings strongly suggest that the less negative electrostatic potential of L. tarentolae leishmanolysin can be the reason for the reduced proteolytic activity detected in this parasite.

Keywords: cloning; comparative modeling; leishmaniasis; molecular dynamics; proteolytic activity.

Figure 1

Unrooted maximum likelihood phylogenetic tree using leishmanolysin protein sequences.L. major, L. braziliensis, L. martiniquensis, and L. tarentolae protein sequences annotated as “leishmanolysin” were obtained from TriTrypDB and were analyzed by Interproscan v5.16-55 to confirm the presence of LSHMANOLYSIN(PR00782) and Peptidase_M8(PF01457) domains. The sequences containing more than 200 residues were aligned and a phylogeny reconstruction based on Maximum Likelihood, Jones-Taylor-Thornton (JTT) was performed and visualized in Dendroscope v3.5.7. Numbers shown at branch nodes are bootstrap values based on 1000 replicates. The closed relation of L. tarentolae and L. major leishmanolysins are indicated, as well as the underlined leishmanolysins selected to be overexpressed.

Figure 2

Three-dimensional structures of L. major (PDB ID: 1LML) and L. tarentolae leishmanolysin 3D-models. The PDB ID of the template and resolution are given in the upper left corner of the upper left quadrant, while the RMSD, coverage degree, and identity between models and the template appear in the upper left corner of the other three. The N-terminal, central region, and C-terminal domains are identified in pink, light blue, and green, respectively. The disulfide bonds are represented by sticks in yellow, the insertion region is identified in dark blue, and the zinc ion appears as a sphere in dark grey.

Figure 3

Differences in the surface electrostatic potential of L. tarentolae and L. major leishmanolysins. The comparative molecular dynamic analysis was performed using the crystal structure of L. major LmjF.10.0460 (PDBid: 1LML) and the modeled L. tarentolae LtaP10.0480, LtaP10.0650, and LtaPcontig00616_1 in pH 5.5 and 7.4. The molecular surface is colored according to electrostatic potential using the Chimera software, where red, white, and blue correspond to acidic, neutral, and basic potentials, respectively.

Figure 4

Proteolytic activity of leishmanolysins from L. tarentolae, L. major and their mutants by Gelatin-SDS-PAGE zymography. The soluble extracts of wild-type L. tarentolae (WT), L. major, and two isolates from each cloned leishmanolysins (LmjF.10.0460 and LtaP10.0650) were submitted to Gelatin-SDS-PAGE electrophoresis. The gels were incubated in 50 mM phosphate buffer pH 5.5 for 72 h at 37 °C. To confirm the enzymatic class, zymograms were incubated in the absence (control) or presence of the ion chelators 1-10-phenanthroline and EDTA at 10 mM and E-64 at 10 µM to exclude cysteine peptidases activity around 63 kDa. The numbers on the left indicate apparent molecular masses of the active bands expressed in kDa.

Figure 5

In solution proteolytic activity of overexpressed L. major LmjF.10.0460 and L. tarentolae LtaP10.0650 mutants. The enzymatic activity was assessed by measuring the hydrolysis of the fluorogenic substrate MCA-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2 at 37 °C for 40 min. Activity of the cloned leishmanolysin from L. tarentolae (Lta + LtaP10.0650) and from L. major (Lta + LmjF.10.0460) were measured in 100 mM glycine-NaOH buffer pH 10.0 buffer the presence or absence of 1 μM1,10-phenantroline (Lta + LtaP10.0650 + phe and Lta + LmjF.10.0460 + phe). Results are expressed as relative fluorescence units (RFU) and the values represent the mean ± standard deviation of three independent experiments performed in triplicate.