In order to gain further insights into the
In order to gain further insights into the role of peptidases in B. xylophilus, four cysteine proteases highly secreted by B. xylophilus (Cardoso et al., 2016) were selected four further characterisation.
Materials and methods
Results and discussion
Structural prediction and analysis The in silico three-dimensional structure suggests that BxCP3 (Fig. 3a) and BxCP11 (Fig. 3b) are pro-enzymes that become active when the pro-peptide is cleaved. The structures of these two proteins were predicted using as templates the crystal structure of human cathepsin L (PDB ID: 1cjl.1.A) for BxCP3 and cathepsin L of Tenebrio molitor (PDB ID: 3qt4.1.A) for BxCP11, according to the best GMQE and QMEAN values obtained after the SWISS-MODEL template library search (Table 2). On the other hand, in silico three-dimensional structure of BxCP7 (Fig. 4a) and BxCP8 (Fig. 4b) were predicted using the crystal structure of cathepsin B from Schistosoma mansoni (PDB ID: 4i04.1A) as template (Table 2) and revealed the presence of a N-terminal pro-peptide and an occluding loop that occludes the active site cleft. This occluding loop unique to cathepsin B proteins, consists of about 20 Puromycin residues and is responsible for the cathepsin B exopeptidase activity in addition to their endopeptidase function (Sajid and McKerrow, 2002). This additional activity is accounted by two histidines of the occluding loop identified in both BxCP7 (H222, H223) and BxCP8 (H179, H180) (Fig. 4), which can function as an additional active center. These histidines serve to anchor the negatively charged carboxylate of the P′2 residue at the C-terminus of the substrates and direct the C-terminal dipeptide into the active site for hydrolysis (Sajid and McKerrow, 2002). Moreover, these histidines have been shown to be linked to the pH dependency of cathepsin B inhibition by its pro-peptide (Quraishi et al., 1999). In addition to the GMQE and QMEAN values (Table 2), the Ramachandran plots validated these structural models with more than 90% of the residues located in the most favoured regions (Supplementary Fig. S1).
Conclusion Four cysteine peptidases, from B. xylophilus secretome, were further characterised using the previously determined cDNA or genomic DNA sequences and bioinformatics approaches. From these, BxCP3 and BxCP11 were identified as cathepsin L-like proteins and BxCP7 and BxCP8 proteins as cathepsin B proteins. Only BxCP8 had high homology with another B. xylophilus cathepsin B present on GenBank database, all the others differ from the closer proteins deposited in this database. In silico three-dimensional structures of BxCP3 and BxCP11 suggest that these proteins, like other cathepsin L-like proteins, are pro-enzymes that become active when the pro-peptide is cleaved. BxCP7 and BxCP8 predicted structures suggested that these are pro-enzymes activated after removal of the N-terminal pro-peptide and revealed the presence of an occluding loop that occludes the active site cleft, typical of cathepsin B proteases. These four cysteine proteases are functional, highly secreted by B. xylophilus and putatively related to its pathogenicity to pine trees. Our findings contribute to increase the knowledge of these essential protein groups and will be supportive for further research on the development of alternative biologic strategies toward this nematode-specific proteases inhibition and B. xylophilus pathogenicity control.
Acknowledgments This research was supported by Foundation for Science and Technology (FCT), Portugal, within the PT2020 Partnership Agreement and COMPETE 2020 under the project UID/BIA/04004/2013 and Instituto do Ambiente, Tecnologia e Vida, Portugal. Joana M.S. Cardoso is funded by post-doctoral fellowship (BPD9) financed by the Project ReNATURE-CENTRO-01-0145-FEDER-000007-Valorization of the Natural Endogenous Resources of the Centro Region, Portugal. Luís Fonseca is funded by post-doctoral fellowship (SFRH/BPD/101325/2014) financed by MEC national funding (Portugal) and by the European Social Fund through POCH (Programa Operacional Capital Humano).