Characterization and classification of five cysteineproteinases expressed by Paragonimus westermani adult worm

Hyun Park,a Suk-Il Kim,b Kyeong-Man Hong,c Mi-Jin Kim,a Chang-Ho Shin,c

Jae-Sook Ryu,d Duk-Young Min,d Jung-Bin Lee,e and Ui Wook Hwangf,*

a Department of Parasitology, College of Medicine, Wonkwang University, Iksan 570-749, Republic of Koreab Department of Parasitology, College of Medicine, Chosun University, Kwangju 501-759, Republic of Koreac Department of Biochemistry, College of Medicine, Wonkwang University, Iksan 570-749, Republic of Koread Department of Parasitology, Hanyang University College of Medicine, Seoul 133-791, Republic of Koreae Department of Forensic Medicine, College of Medicine, Seoul University, Seoul 110-799, Republic of Koreaf Department of Biology, Teachers College, Kyungpook National University, Taegu 702-701, Republic of Korea

Received 1 February 2002; received in revised form 7 January 2003; accepted 19 March 2003

Abstract

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 con-

servation 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 corre-

sponding 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 pro-

teinases.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Paragonimus westermani; Cruzipain-like structure; Cysteine proteinase gene; Phylogenetic relationships

1. Introduction

A trematode Paragonimus westermani causes lung

infection in humans, cats, and tigers that can eat raw

crayfish infected with its metacercariae, which may

Experimental Parasitology 102 (2002) 143–149

www.elsevier.com/locate/yexpr

*Corresponding author. Fax: +82-53-950-6809.

E-mail address: uwhwang@knu.ac.kr (U.W. Hwang).

0014-4894/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S0014-4894(03)00036-5

migrate to the human brain to cause paraplegia, andseizures. The parasite is endemic to Africa, South

America, and Asia including Korea and Japan (Pie-

karski, 1989).

Cysteine proteinases in trematode parasites have been

shown to play important roles in pathogenesis and in-

vasion (McKerrow, 1989). They are reported to hydro-

lyze hemoglobin and collagen, and thus contribute to

the invasion and acquisition of nutrients for the para-sites (Brady et al., 1999; Goldberg et al., 1990; Rosen-

thal et al., 1988). In addition to their pathological roles,

cysteine proteinases have also been suggested to play a

significant role in the reproduction of these parasites. In

P. westermani, an acidic thiol-dependent protease was

reported (Song and Kim, 1994), and a cysteine pro-

teinase gene was also reported from P. westermani

metacercaria (Yamamoto et al., 1994). In our pre-liminary study, we found at least nine proteinases in P.

westermani by gelatin SDS–PAGE. Considerable work

has been done to characterize the cysteine proteinases

from P. westermani, but less information is available

about the classification of cysteine proteinases from

adult P. westermani.

Here, we newly cloned and characterized three cys-

teine proteinase genes (PwCP2, PwCP4, and PwCP5)from adult P. westermani and classified them with pre-

viously reported numerous cysteine proteinases by

phylogenetic analysis.

2. Materials and methods

The metacercariae of P. westermani were obtainedfrom naturally infected crayfish (Cambaroides similis)

collected in Wando-Gun, Korea. Dogs were fed orally

with 150 metacercariae and dissected three months later.

The worms were harvested from cysts in the lungs, and

washed five times with cold saline. Total cellular RNA

and mRNA were purified from the adult worms, and the

isolated mRNA (12 ng) was converted into single-

stranded cDNA using an oligo(dT) 17 primer (Yunet al., 1999). A partial cDNA fragment of the cysteine

proteinase gene was amplified by PCR using degenerate

primers based on the conserved regions of the eukary-

otic cysteine proteinase domains (Eakin et al., 1990).

The primers were dp5 (50-GAR GGI CAR TGY GGITCI TGY TGG-30) and dp3 (50-CCA IGA RTT YTTIAC RAT CCA RTA-30) in which I¼ inosine, R¼A orG, and Y¼T or C.The various cysteine proteinase genes were amplified

from P. westermani using the rapid amplification of

cDNA ends PCR protocol (RACE-PCR) (Park et al.,

1997). Dideoxy nucleotide chain-termination sequencing

(Sanger et al., 1977) was performed on both DNA

strands using a Taq PRISMTM dyeDeoxyTM

sequencing kit (Applied Biosystems). Reactions were

analyzed on a 373 automatic DNA sequencer (AppliedBiosystems) and contiguous sequences were compiled

using MacVectorTM (International Biotechnologies)

sequence analysis software. Note: The nucleotide se-

quence reported in this paper was deposited in the

GenBank under Accession Nos. U56958, U69121, and

U33215 and their classifications in MEROPS database

are as followings: PwCP2 and 4 are orthologues of the

cruzipain (Trypanosoma cruzi) MEROPS ID C01.075and PwCP5 is an orthologue of the cathepsin L (Homo

sapiens) MEROPS ID C01.032.

The amino acid sequences of the cysteine proteinases

were predicted from the nucleotide sequences using

GeneJocky II software (Biosoft). The predicted amino

acid sequences were then multiply aligned with those of

57 cysteine proteinases reported from a variety of or-

ganisms using the Clustal X multiple alignment program(Thompson et al., 1997). Preliminary phylogenetic

analysis was conducted with 57 cysteine proteinases

available from the GenBank using neighbor joining (NJ)

method. Then more detailed phylogenetic trees focused

on PwCP1–5 were reconstructed by both the maximum

parsimony (MP) and NJ methods of PAUP4.0* (Swof-

ford, 2000). In this detailed phylogenetic analysis, we

used a reduced alignment set including only 10 cruzi-pain-like and 10 cathepsin L-like cysteine proteinases as

ingroups and two cathepsin C-like cysteine proteinases

as outgroups.

3. Results and discussion

Three new cysteine proteinase genes, PwCP2(U69121), PwCP4 (U56958), and PwCP5 (U33215) and

the previously reported PwCP1 (U69120) and PwCP3

(U56865) were identified and sequenced from cDNA li-

brary of P. westermani adult worm. As a result of pri-

mary sequence comparisons, it was found that PwCP2

and PwCP4 were most closely related (97.2% identity)

and that PwCP5 had the most divergent amino acid se-

quences among the five characterized in this study, andshared only 18.2% identity with the other four.

Using preliminary phylogenetic analysis conducted

with newly defined three P. westermani cysteine pro-

teinases and 54 cysteine proteinases available from the

GenBank, we recognized that the newly characterized

cysteine proteinases belonged to either a cruzipain-like

group or a cathepsin L-like group shown in NJ tree of

Fig. 1. Overall unrooted NJ tree topology coincidedwith that of Tort et al. (1999) that C1 family peptidases

could be divided into two major evolutionary branches:

branch A including cathepsin B-like and cathepsin C-

like, etc., and branch B including catehepsin L-like,

cruzipain-like, and papain-like, etc.

More detailed phylogenetic analyses focused on the

five P. westermini cysteine proteinases were performed

144 H. Park et al. / Experimental Parasitology 102 (2002) 143–149

Fig. 1. Unrooted neighbor joining tree showing phylogenetic positions of P. westermani cysteine proteinases newly characterized (PwCP2, 4, 5).

PwCP1 P. westermani cysteine proteinase (GenBank Accession No., U69120); PwCP2 P. westermani cysteine proteinase (U69121); PwCP3 P.

westermani cysteine proteinase (U56865); PwCP4 P. westermani cysteine proteinase PwCP5 P. westermani cysteine proteinase (U33215); Pw28CCP,

P. westermani 28-kDa cruzipain-like cystein protease (U70537); NTP, P. westermani neutral thiol protease (D21124); CsCP, Clonorchis sinensis

cysteine proteinase 1 precursor (AF093243); FhCL, Fasciola hepatica cathepsin L (AB009306); FhCL2, F. hepatica secreted cathepsin L2 (U62289);

FhCP, F. sp. cysteine protease (S70380); SmCL1, S. mansoni Puerto preprocathepsin L1 (U07345); SmCL2, S. mansoni (Liberian) cathepsin L

(Z32529); SmCC, S. mansoni (Liberian) cathepsin C (Z32531); Sm31, S. mansoni cathepsin B (M21309); SjCL2, S. japonicum preprocathepsin L

(U38476); SjCC, S. japonicum preprocathepsin C (U77932); Smcal, S. mansoni calpain (M74233); Sm32, S. mansoni hemoglobinase (M21308); SjCB,

S. japonicum cathepsin B (X70968); Sj32, S. japonicum Sj32 (hemoglobinase) (X70967); SeCL, Spirometra erinacei cysteine proteinase (D63670);

AcCP, Ancylostoma caninum (AcCP-1) cathepsin B (U18911); CeCB, Caenorhabditis elegans (Bristol N2) cathepsin B (L39927); HcCP1,Haemonchus

contortus cysteine proteinase (HMCP1) (Z69342); HcCP2, H. contortus cysteine proteinase (HMCP2) (Z69343); HcCP4, H. contortus cysteine

proteinase (HMCP4) (Z69345); HcCP5, H. contortus cysteine proteinase (HMCP5) (Z69346); HcCP6, H. contortus cysteine proteinase (Z81327);

TvPt, Trichomonas vaginalis putative cysteine protease, partial (X70823); GmCP, Giardia muris cysteine protease (GMCP1) (AF006198); GiCP1, G.

intestinalis cysteine protease (U83277); GiCP2 G. intestinalis cysteine protease (U83275); TcCZ1, Toxocara canis cathepsin Z1 (AF143817); TcCL, T.

canis cathepsin L (U53172); TcCB, T. cruzi cathepsin B (AF043246); TcCP, Trypanosome cysteine protease (X54353); Tc, T. cruzi cruzain (M84342);

TcCP, T. congolense cysteine protease (L25130); LdCP, Leishmania donovani chagasi promastigote-specific cysteine protease (AF004593); LmCL,

Leishmania major cathepsin L (U43706); LmCB, Leishmania major cathepsin B (U43705); moCL, mouse cathepsin L (M20495); huCL, human

cathepsin L (M20496); GCCB, human cathepsin B (gastric) (L16510); huCB, human cathepsin B (kidney) (M14221); huCS, human cathepsin S

(CTSS) (M90696); huCS, human cathepsin S (alveolar macrophage) (M86553); huCX, human cathepsin X precursor (ovary) (AF073890); boCX,

human cathepsin X mRNA (osteoclastoma) (U20280); papain, Carica papaya papain (M15203); plCP, Zea mays cysteine protease (mir1)

(AF019145); chymopapain, C. papaya chymopapain (X97789); HgCL, Heteroderma glycines cathepsin L (nematodes) (Y09498); bromelain pt, plant,

Ananas comosus bromelain (D14057).

H. Park et al. / Experimental Parasitology 102 (2002) 143–149 145

using two tree reconstructing methods (MP and NJ)with a reduced alignment set including only 22 cysteine

proteinases which belonged to the cruzipain-like, ca-

thepsin L-like, and cathepsin C-like groups (Fig. 2). The

results showed that PwCP1, 2, 3, and 4 were grouped

with the cruzipain-like cysteine proteinases (bootstrap-

ping values 97 and 100% in MP and NJ trees, respec-

tively) and that PwCP5 with the cathepsin L-like

cysteine proteinases (76 and 100%), as shown in Fig. 2.These results were strongly supported by high boot-

strapping values in both the MP and NJ trees as men-

tioned. The MP and NJ trees were identical in overall

tree topology but have slightly different bootstrapping

values. Within the clade of cruzipain-like cysteine pro-

teinases, PwCP2 and 4 were most closely related to each

other, as expected from their high sequence similarity.

PwCP5 of cathepsin L-like is the first non-cruzipain-likecysteine proteinase reported from P. westermani con-

sidering that all the P. westermani cysteine proteinases

known so far belong to the cruzipain-like group. Until

now, Schistosoma mansoni and Schistosoma japonicum

were the only organisms from which two different genes

encoding both the cruzipain (SmCL1, SmCJ1) and ca-

thepsin L-like (SmCL2, SmCJ2) proteinases have been

isolated and characterized, although the research toobtain functionally active recombinant enzymes was

done only in S. mansoni (Brinkworth et al., 2000). Thus,

the present study is worthy of reporting one another

case, the third organism, which has the two kinds of

genes encoding both the cruzipain and cathepsin L-like

cysteine proteinases. It is also quite intriguing that all

the three organisms known so far are trematodes. It

implies that possession of both the cruzipain and ca-thepsin L-like cysteine proteinases in an organism might

not be rare phenomena in trematodes.

It was known that cysteine proteinases of C1 pepti-

dase family have highly conserved structure (Brady

et al., 2000; Brinkworth et al., 2000; Tort et al., 1999).

For instances, they consists of two domains, NH2-ter-

minal half (helical structure domain) and COOH-ter-

minal half (predominantly b-sheet structure domain).Generally, there are three conserved disulfide bridges for

forming appropriate and stable tertiary structures, two

in the first NH2-domain and one in the second COOH-

domain. The sites of the six cysteine residues related

with three disulfide bonds are shown in Fig. 3 and the

sites are completely conserved with cysteine residues in

all the examined cysteine proteinases including

PwCP1–5. It shows that the PwCP1–5 could be foldedsuccessfully into functional enzymes.

Recently, Brinkworth et al. (2000) has reported six

cruzipain signature residues (Asn 33, Trp 38, Ala 124,

Leu 127, Leu 164, and Pro 174, according to amino acid

residue numbering of cysteine proteinase of T. cruzi)

through comparison of primary structures of cysteine

proteinases between cruzipain and cathepsin L-like

groups. Four (PwCP1–4) of five P. westermini cysteine

proteinases focused on this study which belong to cru-

zipain group have the exact cruzipain signature se-

Fig. 2. Most parsimonious tree inferred from amino acid sequences of

22 cysteine proteinases including PwCP1–5. SmCC and SjCC were

used as outgroups. The numbers on the tree above branches indicate

bootstrapping values obtained with 100 replicates. Tree-bisection-re-

connection (TBR) method was used as a branch-swapping algorithm.

The numbers in parentheses are bootstrapping values obtained from

NJ analysis. In MP analysis, all characters had equal weighting. Of 471

total characters, 55 are constant, 128 variable characters are parsi-

mony-uninformative, and 288 are parsimony-informative. Tree length

¼ 2208, consistency index (CI) ¼ 0.7785, retention index (RI) ¼0.6823, rescaled consistency index ¼ 0.5312. Cathepsin L-like cysteine

proteinases formed a strong monophyletic group with PwCP1–5 in the

three phylogenetic trees. PwCP1 P. westermani cysteine proteinase

(GenBank Accession No., U69120); PwCP2 P. westermani cysteine

proteinase (U69121); PwCP3 P. westermani cysteine proteinase

(U56865); PwCP4 P. westermani cysteine proteinase PwCP5 P. west-

ermani cysteine proteinase (U33215); PwCP, P. westermani cysteine

proteinase (AF071801); PwNTP, P. westermani neutral thiol protease

(D21124); Pw28CCP, P. westermani 28-kDa a cruzipain-like cysteine

protease (U70537); CsCP1, C. sinensis cysteine proteinase (AB020036);

CsCP2, C. sinensis cysteine proteinase 1 precursor (AF093243);

SmCL1, S. mansoni puerto rican preprocathepsin L1 (U07345);

huCS1, human cathepsin S (CTSS) (M90696); huCS2, human ca-

thepsin S (alveolar macrophage) (M86553); boCX, human cathepsin X

mRNA (osteoclastoma) (U20280); HgCL, nematode heteroderma

glycines cathepsin L (Y09498); moCL, mouse cathepsin L (M20495);

huCL, human cathepsin L (M20496); SeCL, S. erinacei cysteine pro-

teinase (D63670); SmCL2, S. mansoni cathepsin L (Z32529); SjCL, S.

japonicum preprocathepsin L (U38476); SmCC, S. mansoni (Liberian)

cathepsin C (Z32531); SjCC, S. japonicum preprocathepsin C

(U77932).

146 H. Park et al. / Experimental Parasitology 102 (2002) 143–149

Fig. 3. Deduced amino acid sequence alignment of 12 cysteine proteinases including three cysteine proteinase genes (PwCP2, 4, and 5) newly defined

from P. westermani. PwCP1 P. westermani cysteine proteinase (GenBank Accession No., U69120); PwCP2 P. westermani cysteine proteinase

(U69121); PwCP3 P. westermani cysteine proteinase (U56865); PwCP4 P. westermani cysteine proteinase (U56958); PwNTP, P. westermani neutral

thiol protease (D21124); PwCP, P. westermani cysteine proteinase (AF071801); Pw28CCP, P. westermani 28-kDa cruzipain-like cysteine protease

(U70537); SmCL1, S. mansoni Puerto rican preprocathepsin L1 (U07345); CsCP1, C. sinensis cysteine proteinase (AB020036); PwCP5 P. westermani

cysteine proteinase (U33215); huCL, human cathepsin L (M20496); SmCL2, S. mansoni cathepsin L (Z32529). Total alignment length ¼ 441; ‘‘-’’

missing data or alignment gap; ‘‘?’’ unidentified amino acid sequence; ‘‘.’’ same amino acid residue with that of the first line (PwCP1) of the

alignment. The names of the enzymes belonging to the cathepsin L-like are marked in italic letters, CsCP1, PwCP5, huCL, and SmCL2. White boxes

indicate conserved cystein residues related with forming disulfide bridges and gray boxes six cruzipain signature residues which correspond to Asn 33,

Trp 38, Ala 124, Leu 127, Leu 164, and Pro 174 (in order) reported by Brinkworth et al. (2000) according to a residue numbering of cystein proteinase

of T. cruzi. Bold letters mark cathepsin L-like signature residues suggested firstly here. White arrow indicates the fifth cruzipain signature residue

(Leu) showing which may be inappropriate as a cruzipain signature residue because of high variability among cruzipain proteinases. ‘‘�’’ indicates a

processing site: the first one is for removal of the signal peptide and the second one is for cleavage of the proregion peptide from the mature enzyme.

‘‘}’’ and ‘‘N’’ indicate S3 pocket position and two- or three-residue insertion site of S3 subsite causing difference of substrate specificity betweenSmCL1 and SmCL2 (Brady et al., 2000). The ERFNIN motif region (Karrer et al., 1993) is marked with asterisks above the relevant residues in the

proregion peptides.

H. Park et al. / Experimental Parasitology 102 (2002) 143–149 147

quences in five sites corresponding to Asn 33, Trp 38,Ala 124, Leu 127, and Pro 174, except that the last Pro

174 residue was unidentified in PwCP2 because of se-

quence incompleteness (Fig. 3). However, the fifth cru-

zipain signature site (Leu 164) was not conserved, and

thus except for leucine in SmCL1 and PwCP1, threonine

in PwCP2–4, PwNTP, PwCP, and Pw28CCP and serine

in PwCP3 were found in the corresponding site (Fig. 3).

In P. westermini cysteine proteinases, seven among eightreported so far have not a hydrophobic leucine residue

but one of two hydrophilic residues (threonine or serine)

except PwCP1 as shown in Fig. 3 (white arrow). This is

likely to show that the fifth one (Leu 164) may be in-

appropriate as a cruzipain signature residue. PwCP5

considered as cathepsin L-like group based on the

phylogenetic analysis did not have any cruzipain signa-

ture residues.Through comparison of all kinds of cysteine pro-

teinases published until now, we carefully looked for

signature residues specific to cathepsin L-like group and

thus useful to identify it as in the cruzipain group. As a

result, we found four signature candidate sites (Gly 174,

Asn 175, Ala 325, and Pro 331—human cathepsin L

numbering) for cathepsin L-like group, which are con-

served within cathepsin L-like group and also differentfrom those of cruzipain and other cysteine proteinase

groups. Unfortunately, existences of Ala 325 and Pro

331 in PwCP5 were impossible to confirm because of 30-end incomplete sequencing. Except for the two, PwCP5

with other cathepsin L-like cysteine proteinases have the

two remaining residues (Gly 174 and Asn 175 high-

lighted with bold letters in Fig. 3) considered as the most

important signature residues characteristic of cathepsinL-like proteinases. These two possible signature resi-

dues, Gly 174 (this residue does not exist in some ca-

thepsin L-like cysteine proteinases) and (the most

conserved signature residue), seem to have more im-

portant meaning than the other two to distinguish ca-

thepsin L-like cysteine proteinases from cruzipain

group, because the two are corresponding to the S3

pocket position (Asn 175) or its vicinal position (Gly174) related with difference of substrate specificity be-

tween SmCL1 (cruzipain group) and SmCL2 (cathepsin

L-like) (Brady et al., 2000). According to Brady et al.

(2000), such substrate specificity difference between the

two are caused mainly by the two-residue insertion

(hydrophilic residues, Tyr–Gly) shown in immediate

upstream position of the S3 pocket of SmCL2. In the

equivalent position, PwCP1–4 belonging to cruzipaingroup have no insertion residues as SmCL1 and other

cruzipain cysteine proteinases, whereas PwCP5 has

three-residue insertion (hydrophilic residues, Ser–Tyr–

Gly) as SmCL2 and other cathepsin L-like cysteine

proteinases. Compared to the two-residue insertion in

SmCL2, the three-residue insertion appeared in PwCP5

may bring bigger difference in substrate specificity be-

tween PwCP1–4 and PwCP5 than that observed betweenSmCL1 and SmCL2 (Brady et al., 2000). Such pre-

sumption is quite plausible considering extremely lower

amino acid sequence similarity (18.2%) between

PwCP1–4 and PwCP5 (44% between SmCL1 and

SmCL2).

Yun et al. (2000) reported an extensive work for

exploring 28-kDa cruzipain-like cysteine protease

(Pw28CCP) from P. westermani. According to their re-sults, Pw28CCP is located in the intestinal epithelium

and its recombinant protein expressed in Escherichia coli

appeared highly specific and sensitive antigenicity with

sera from patients with active paragonimiasis. Our

present result showed that two of three newly charac-

terized cysteine proteinases from P. westermani be-

longed to the cruzipain-like group. As shown in Fig. 2,

PwCP1–4 are very closely related with PW28CCP withinthe cruzipain-like group. It suggests that most of P.

westermani cysteine proteinases may be cruzipain-like

one closely related with Pw28CCP. The fact that most of

cysteine proteinases from P. westermani reported so far

are cruzipain-like types implies strongly that a new

powerful drug for paragonimiasis could be designed and

developed if we focus on exploration of anti-agents

against P. wesetrmani cruzipain-like cysteine protein-ases.

Acknowledgments

This study was supported by a grant from the Min-

istry of Education for Basic Medical Science (KRF-

1997-021-F00275), by Wonkwang University (2001), in

part, and by research fund from Chosun University in

2000. This work was also partly supported by the KoreaResearch Foundation grant (KRF-2002-015-CS0033) to

U.W.H.

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