Proteases have been divided into groups on
Proteases have been divided into groups on the basis of the catalytic mechanism used during the hydrolytic process. The main catalytic types are serine, threonine, aspartate, metallo and cysteine proteases. The first cysteine protease, named papain, was purified and characterised from Carica papaya, the papaya fruit, and was also the first cysteine protease structure to be solved. Since its discovery, numerous proteases that have sequences in common with papain have been called papain-like enzymes, also named cathepsins. Papain-like, or Clan CA proteases, are further divided into prmt5 inhibitor and the most common and important parasite proteases cathepsin L-like and cathepsin B belong to family C1 (Sajid and McKerrow, 2002). The main differences between these two subfamilies rely on the sequence of the pro-peptide and its length (Turk et al., 2012). The presence of cysteine proteases in B. xylophilus have been previously reported in genomic and EST (Kang et al., 2009, Kikuchi et al., 2011) studies, but no further studies on the identified cysteine proteases have been performed. According to the data in GenBank database (Benson et al., 2013), several sequences of cathepsin L-like proteins from plant-parasitic nematodes have been reported such as cathepsin L-like sequences from B. xylophilus (ACH69776; ACH56225), B. mucronatus (AID50178), Ditylenchus destructor (ACT35690), Radopholus similis (ACH56226), Rotylenchulus reniformis (AAY45870) and from different species of the genera Globodera (29 cathepsin L-like sequences), Heterodera (7) and Meloidogyne (6). However, many of these sequences are partial sequences, some of them representing just a small fragment of the complete sequence. On the other hand, cathepsin B have rarely been studied and from plant-parasitic nematodes only one sequence from B. xylophilus (ACZ13346) and other from R. similis (ADK46902) was found in GenBank.
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 amino acids 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).