Three new members of the
cysteine proteinase gene family of Paragonimus westermani have been isolated and classified. Comparisons of the predicted amino acid sequences of PwCP2 (U69121), PwCP4 (U56958), and PwCP5 (U33215) were performed with those of the previously reported PwCP1 (U69120) and PwCP3 (U56865) sequence. The
amino acid alignment showed conservation of the
cysteine,
histidine, and
asparagine residue that form the catalytic triad. With 57
cysteine proteinases including PwCP1-5, we conducted phylogenetic analysis using neighbor joining method (NJ). A resultant unrooted tree revealed that PwCP1-5 were clustered with
cruzipain-like or
cathepsin L-like
cysteine proteinases. More detailed phylogenetic analyses with a reduced alignment set (22
cysteine proteinases) were performed by NJ and maximum parsimony (MP) methods. The results showed coincidently that PwCP1, 2, 3, and 4 belonged to the group of previously reported
cruzipain-like
cysteine proteinases (bootstrapping values of 97 and 100% in the MP and NJ trees) but PwCP5 to
cathepsin L-like
cysteine proteinases (the value of 76 and 100% in MP and NJ trees). Within the
cruzipain-like clade, PwCP2 and 4 were found to be the most closely related. PwCP 2, 3, and 4 have five of six
cruzipain signature sequences known previously, whereas PwCP5 do not have any
cruzipain sequences in the corresponding sites. We found that two signature candidate sites (Gly 174, Asn 175--human
cathepsin L numbering) for
cathepsin L-like group are conserved in PwCP5, which are conserved within
cathepsin L-like group and also different from those of
cruzipain and other
cysteine proteinase groups. PwCP5 has three-residue insertion (hydrophilic residues, Ser-Tyr-Gly) within the position corresponding to S3 subsite of SmCL2. Compared to the two-residue insertion (Tyr-Gly) in SmCL2, the three-residue insertion appeared in PwCP5 may bring bigger difference in substrate specificity between PwCP1-4 (
cruzipain) and PwCP5 (
cathepsin L-like). Such presumption is quite plausible considering extremely lower amino acid sequence similarity (18.2%) between PwCP1-4 and PwCP5. The present study is worthy of reporting one another case, the third organism after Schistosoma mansoni and Schistosoma japonicum, which has the two kinds of genes encoding both the
cruzipain and
cathepsin L-like
cysteine proteinases. In addition, the fact that most of
cysteine proteinases from P. westermani are
cruzipain-like type implies strongly that a new powerful
drug for
paragonimiasis could be designed and developed if we focus on the exploration of anti-agents against P. westermani
cruzipain-like
cysteine proteinases.