Suk-Il Kim

CHARACTERIZATION AND LARGE-SCALE EXPRESSION OF THE RECOMBINANT CYSTEINE PROTEINASE FROM ADULT CLONORCHIS SINENSIS

Cysteine proteinases play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based approach of drug design. As the first step toward applying this strategy to design inhibitors as antiparasitic agents for Clonorchis sinensis, we overexpressed and characterized the 24-kDa cysteine proteinase from adult worms. First, the partial cysteine proteinase gene from C. sinensis was cloned by performing reverse transcription polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5′ and the 3′ regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends–polymerase chain reaction (RACE-PCR). The cDNA has an open reading frame of 981 bp, and the deduced amino acid sequence shares similarity with the cathepsin L–like cysteine proteinases from Schistosoma mansoni, Paragonimus westermani metacercaria, Fasciola hepatica, and human cathepsin L by 52%, 47%, 34%, and 29%, respectively. The cysteine proteinase was then overexpressed in the yeast Pichia pastoris as an active enzyme on a large-scale basis (19.7 mg/L). The active recombinant enzyme was purified from culture media using a Ni2+–NTA–agarose affinity column and gel filtration chromatography. This 24-kDa recombinant protein exhibited a substrate preference for Z-Phe-Arg-AMC (benzyloxycarbonyl-l-phenylalanyl-l-arginine-7-amino-4-methyl-coumarin) compared with Z-Arg-Arg-AMC, and the activity was inhibited by E-64 (l-trans-epoxysuccinylleucylamido(4-quanidino)butane).

Identification and characterization of Paragonimus westermani leucine aminopeptidase

Paragonimus westermani is a tissue-invading trematode parasite that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals. While aminopeptidases play important roles in trematodes in the catabolism of host hemoglobin, an essential source of nutrient for the parasite, little is known about aminopeptidase in Paragonimus. Presently, we isolated a cDNA encoding a 58 kDa P. westermani leucine aminopeptidase (PwLAP). Deduced amino acid sequence of PwLAP exhibited significant sequence homology with LAP from Schistosoma spp. and Fasciola hepatica. Biochemical analysis of the recombinant PwLAP protein demonstrated preferential substrate specificity for Leu-NHMec and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of leucine aminopeptidase. PwLAP exhibited relatively higher enzyme activity in the presence of Mn2+ compared to Schistosoma mansoni LAP. Based on the biochemical properties and immunohistochemical analysis, PwLAP is concluded to represent a leucine aminopeptidase. The enzyme is most likely responsible for the catabolism of host hemoglobin, and, hence, represents a potential target of Paragonimus chemotherapy.

Characterization and classification of five cysteine proteinases expressed by Paragonimus westermani adult worm.

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.

Mucous cell differentiation in a unicystic ameloblastoma

The authors report a case of unicystic ameloblastoma with mucous cell differentiation in the right mandible of a 24-year-old Korean male who suffered from painful swelling for 2 months. A radiograph showed a well-circumscribed radiolucent lesion between the root of the right first premolar and the first molar tooth. Microscopic examination revealed the cystic lesion was lined with ameloblastic epithelium and goblet cells in the epithelium. The mucous cells reacted positively to mucicarmine stain. The possible pathogenic mechanism of this case reflects the pluripotential character of the odontogenic epithelium. The prognosis is probably that expected for conventional unicystic ameloblastoma.