A group of cathepsins L as predominant proteolytic enzymes of Eudiplozoon nipponicum

Cathepsin L-like peptidases of the hematophagous monogenean Eudiplozoon nipponicum (family Diplozoidae) seem to play a key role in the digestion of host blood proteins. We have identified at least 13 different transcripts coding for cathepsins L in the transcriptome of adult worms. Two of them, the most transcribed ones, named EnCL1 [GenBank: KP793605] and EnCL3 [GenBank: KP793606], were heterologously expressed in E. coli bacterial (rEnCL1b) and P. pastoris yeast systems (rEnCL1y and rEnCL3y). While rEnCL3y was obtained as a relatively stable zymogen (proenzyme) and could be autoactivated at low pH, rEnCL1y undertook self-processing in yeast medium and was very unstable. Both recombinant enzymes exhibited substrate preferences characteristic for cathepsin L-like peptidases, and were able to hydrolyze hemoglobin, albumin, type I collagen, IgG, and fibrinogen. Specific RNA probes and monospecific antibodies localized the transcripts/enzymes inside the vesicles of haematin cells of the digestive tract. Moreover, both EnCL1 and EnCL3 were also detected in the gut lumen of adult worms. These results strongly support the idea that both CL endopeptidases studied participate in processing of blood. Additionally, bioinformatic sequence analyses were performed in order to compare the other cathepsin L-like sequences found in the transcriptome of adult E. nipponicum. One of the sequences (named EnCL2) has a conspicuously divergent composition of the S2 subsite, and could be expected to efficiently cleave the substrates with proline in P2 position, including the repeated Gly-Pro-Xaa motifs occurring within the amino acid sequence of collagen. Thanks to this, EnCL2 might participate in disruption of host tissues, e.g. of gill lamellae.