Jung-Mi Kang

A family of cathepsin F cysteine proteases of Clonorchis sinensis is the major secreted proteins that are expressed in the intestine of the parasite.

Cysteine proteases of helminth parasites play essential roles in parasite physiology as well as in a variety of important pathobiological processes. In this study, we identified a multigene family of cathepsin F cysteine proteases in Clonorchis sinensis (CsCFs). We identified a total of 12 CsCF genes through cDNA cloning using degenerate PCR primers followed by RACE. Sequence and phylogenetic analysis of the genes suggested they belonged to the cathepsin F-like enzyme family and further clustered into three different subfamilies. Enzymatic and proteomic analysis of C. sinensis excretory and secretory products (ESP) revealed that multiple isoforms of CsCF were the major proteins present in the ESP and the proteolytic activity of the ESP is mainly attributable to the enzymes. Comparative analysis of representative enzymes for each subfamily, CsCF-4, CsCF-6, and CsCF-11, showed that they share similar biochemical properties typical for cathepsin F-like enzymes, but significant differences were also identified. The enzymes were expressed throughout various developmental stages of the parasite and the transcripts increased gradually in accordance with the maturation of the parasite. Immunolocalization analysis of CsCFs showed that they were mainly localized in the intestine and intestinal contents of the parasite. These results collectively suggested that CsCFs, which are apparently synthesized in the epithelial cells lining the parasite intestine and secreted into the intestinal lumen of the parasite, might have a cooperative role for nutrient uptake in the parasite. Furthermore, they were eventually secreted into outside of the parasite and may perform additional functions for host-parasite interactions.

Genetic polymorphism and effect of natural selection at domain I of apical membrane antigen-1 (AMA-1) in Plasmodium vivax isolates from Myanmar

Malaria is endemic or hypoendemic in Myanmar and the country still contributes to the high level of malaria deaths in South-East Asia. Although information on the nature and extent of population diversity within malaria parasites in the country is essential not only for understanding the epidemic situation but also to establish a proper control strategy, very little data is currently available on the extent of genetic polymorphisms of the malaria parasites in Myanmar. In this study, we analyzed the genetic polymorphism and natural selection at domain I of the apical membrane antigen-1 (AMA-1) among Plasmodium vivax Myanmar isolates. A total of 34 distinguishable haplotypes were identified among the 76 isolates sequenced. Comparison with the previously available PvAMA-1 sequences in the GenBank database revealed that 21 of them were new haplotypes that have never been reported till date. The difference between the rate of nonsynonymous (dN) and synonymous (dS) mutations was positive (dN-dS, 0.013+/-0.005), suggesting the domain I is under positive natural selection. The Tajimas D statistics was found to be -0.74652, suggesting that the gene has evolved under population size expansion and/or positive selection. The minimum recombination events were also high, indicating that recombination may occur within the domain I resulting in allelic diversity of PvAMA-1. Our results collectively suggest that PvAMA-1 displays high genetic polymorphism among Myanmar P. vivax isolates with highly diversifying selection at domain I. These results have significant implications in understanding the nature of P. vivax population circulating in Myanmar as well as providing useful information for malaria vaccine development based on this antigen.

Genetic diversity and natural selection of Duffy binding protein of Plasmodium vivax Korean isolates

Plasmodium vivax Duffy binding protein (PvDBP) is a micronemal type I membrane protein that plays an essential role in erythrocyte invasion of merozoites. PvDBP is a prime blood stage vaccine candidate antigen against P. vivax, but its polymorphic nature represents a major obstacle to the successful design of a protective vaccine against vivax malaria. In this study, we analyzed the genetic polymorphism and natural selection at the N-terminal cysteine-rich region of PvDBP (PvDBPII) among 70 P. vivax isolates collected from Korean patients during 2005-2010. Seventeen single nucleotide polymorphisms (SNP), which resulted in 14 non-synonymous and 3 synonymous mutations, were found in PvDBPII among the Korean P. vivax isolates. Sequence analyses revealed that 13 different PvDBPII haplotypes, which were clustered into 3 distinct clades, were identified in Korean P. vivax isolates. The difference between the rates of nonsynomyous and synonymous mutations suggested that the region has evolved under natural selection. High selective pressure preferentially acted on regions identified or predicted to be B- and T-cell epitopes and MHC binding regions of PvDBPII. Recombination may also contribute to genetic diversity of PvDBPII. Our results suggest that PvDBPII of Korean P. vivax isolates display a limited genetic polymorphism and are under selective pressure. These results have significant implications for understanding the nature of the P. vivax population circulating in Korea and provide useful information for development of malaria vaccines based on this antigen.

Genetic polymorphism and natural selection in the C-terminal 42 kDa region of merozoite surface protein-1 among Plasmodium vivax Korean isolates

BackgroundThe carboxy-terminal 42 kDa region of Plasmodium vivax merozoite surface protein-1 (PvMSP-142) is a leading candidate antigen for blood stage vaccine development. However, this region has been observed to be highly polymorphic among filed isolates of P. vivax. Therefore it is important to analyse the existing diversity of this antigen in the field isolates of P. vivax. In this study, the genetic diversity and natural selection in PvMSP-142 among P. vivax Korean isolates were analysed.MethodsA total of 149 P. vivax- infected blood samples collected from patients in Korea were used. The region flanking PvMSP-142 was amplified by PCR, cloned into Escherichia coli, and then sequenced. The polymorphic characteristic and natural selection of PvMSP-142 were analysed using the DNASTAR, MEGA4 and DnaSP programs.ResultsA total of 11 distinct haplotypes of PvMSP-142 with 40 amino acid changes, as compared to the reference Sal I sequence, were identified in the Korean P. vivax isolates. Most of the mutations were concentrated in the 33 kDa fragment (PvMSP-133), but a novel mutation was found in the 19 kDa fragment (PvMSP-119). PvMSP-142 of Korean isolates appeared to be under balancing selection. Recombination may also play a role in the resulting genetic diversity of PvMSP-142.ConclusionsPvMSP-142 of Korean P. vivax isolates displayed allelic polymorphisms caused by mutation, recombination and balancing selection. These results will be useful for understanding the nature of the P. vivax population in Korea and for development of a PvMSP-142 based vaccine against P. vivax.

Genetic polymorphism and natural selection of Duffy binding protein of Plasmodium vivax Myanmar isolates

BackgroundPlasmodium vivax Duffy binding protein (PvDBP) plays an essential role in erythrocyte invasion and a potential asexual blood stage vaccine candidate antigen against P. vivax. The polymorphic nature of PvDBP, particularly amino terminal cysteine-rich region (PvDBPII), represents a major impediment to the successful design of a protective vaccine against vivax malaria. In this study, the genetic polymorphism and natural selection at PvDBPII among Myanmar P. vivax isolates were analysed.MethodsFifty-four P. vivax infected blood samples collected from patients in Myanmar were used. The region flanking PvDBPII was amplified by PCR, cloned into Escherichia coli, and sequenced. The polymorphic characters and natural selection of the region were analysed using the DnaSP and MEGA4 programs.ResultsThirty-two point mutations (28 non-synonymous and four synonymous mutations) were identified in PvDBPII among the Myanmar P. vivax isolates. Sequence analyses revealed that 12 different PvDBPII haplotypes were identified in Myanmar P. vivax isolates and that the region has evolved under positive natural selection. High selective pressure preferentially acted on regions identified as B- and T-cell epitopes of PvDBPII. Recombination may also be played a role in the resulting genetic diversity of PvDBPII.ConclusionsPvDBPII of Myanmar P. vivax isolates displays a high level of genetic polymorphism and is under selective pressure. Myanmar P. vivax isolates share distinct types of PvDBPII alleles that are different from those of other geographical areas. These results will be useful for understanding the nature of the P. vivax population in Myanmar and for development of PvDBPII-based vaccine.

Identification and functional characterization of CsStefin-1, a cysteine protease inhibitor of Clonorchis sinensis

Cathepsin Fs of Clonorchis sinensis (CsCFs) are major secreted proteins that are expressed in the intestine of the parasite and play pivotal roles in parasite nutrition and host-parasite interactions. However, strict regulation of their activities is also essential to minimize inadequate superfluous damage to the parasite and host. In this study, we identified and characterized a novel cysteine protease inhibitor of C. sinensis, CsStefin-1, as a modulator of CsCFs. CsStefin-1 was shown to be a typical cysteine protease inhibitor of family 1 cystatins that lacks the N-terminal signal peptide and C-terminal cysteine residues required for disulfide bond formation. Phylogenetic and structural analyses also showed that CsStefin-1 is a family 1 intracellular cystatin. Bacterially expressed CsStefin-1 effectively inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, papain, and CsCFs. CsStefin-1 was active over a wide pH range and was highly stable under physiological conditions. CsStefin-1 also inhibited the processing of CsCFs. CsStefin-1 was expressed throughout various developmental stages of the parasite from metacercaria to adult worm and the protein was detected in worm extract, but not in the excretory and secretory products of adult worm. Immunolocalization analysis showed that CsStefin-1 was mainly localized to the intestinal epithelium, where CsCFs are actively synthesized. Our results collectively suggest the regulatory functions of CsStefin-1, modulation of CsCFs activity and processing, to protect the parasite from superfluous damage by the endogenous cysteine proteases.

Identification and characterization of a serine protease inhibitor of Clonorchis sinensis

A gene encoding a serine protease inhibitor of Clonorchis sinensis (CsSERPIN) was identified and characterized. CsSERPIN contained an open reading frame of 1158bp that encoded 385 amino acid residues. Sequence analysis of the primary structure of CsSERPIN revealed that it had essential structural motifs including a reactive central loop (RCL), which well conserved in the serine protease inhibitor (serpin) superfamily. CsSERPIN was classified as a member of the ovalbumin-type serpin family on the basis of phylogenetic analysis and the absence of a classical N-terminal signal peptide. Recombinant CsSERPIN showed an inhibitory effect on chymotrypsin in a dose-dependent manner, but did not effectively inhibit trypsin, thrombin, elastases or cathepsin G. Optimal pH values of CsSERPIN were between 7.0 and 9.0, as evidenced by the rapid loss of inhibitory activity under acidic conditions. CsSERPIN was expressed at various developmental stages of the parasite, from eggs to adult worms, but its expression level was higher in eggs and adult worms than in metacercariae and juvenile worms. CsSERPIN was identified in the soluble extract of the parasite, but not in the excretory and secretory products (ESP) or insoluble extract of the parasite. Immunolocalization analysis of CsSERPIN showed that it mainly localized to the eggs and vitelline glands of the adult worm. These results suggest that intracellular CsSERPIN may be possibly involved in maintaining the physiology of eggs as well as in egg production of C. sinensis by regulating endogenous serine proteases.

Molecular cloning and characterization of a paramyosin from Clonorchis sinensis.

Paramyosin is a myofibrillar protein present in helminth parasites and plays multifunctional roles in host-parasite interactions. In this study, we identified the gene encoding paramyosin of Clonorchis sinensis (CsPmy) and characterized biochemical and immunological properties of its recombinant protein. CsPmy showed a high level of sequence identity with paramyosin from other helminth parasites. Recombinant CsPmy (rCsPmy) expressed in bacteria had an approximate molecular weight of 100 kDa and bound both human collagen and complement 9. The protein was constitutively expressed in various developmental stages of the parasite. Imunofluorescence analysis revealed that CsPmy was mainly localized in the tegument, subtegumental muscles, and the muscle layer surrounding the intestine of the parasite. The rCsPmy showed high levels of positive reactions (74.6%, 56/75) against sera from patients with clonorchiasis. Immunization of experimental rats with rCsPmy evoked high levels of IgG production. These results collectively suggest that CsPmy is a multifunctional protein that not only contributes to the muscle layer structure but also to non-muscular functions in host-parasite interactions. Successful induction of host IgG production also suggests that CsPmy can be applied as a diagnostic antigen and/or vaccine candidate for clonorchiasis.

Cryptostatin, a chagasin-family cysteine protease inhibitor of Cryptosporidium parvum

Cysteine proteases of pathogenic protozoan parasites play pivotal roles in the life cycle of parasites, but strict regulation of their activities is also essential for maintenance of parasite physiology and interaction with hosts. In this study, we identified and characterized cryptostatin, a novel inhibitor of cysteine protease (ICP) of Cryptosporidium parvum. Cryptostatin showed low sequence identity to other chagasin-family ICPs, but 3 motifs (NPTTG, GXGG, and RPW/F motifs), which are evolutionarily conserved in chagasin-family ICPs, were found in the sequence. The overall structure of cryptostatin consisted of 8 β-strands that progressed in parallel and closely resembled the immunoglobulin fold. Recombinant cryptostatin inhibited various cysteine proteases, including papain, human cathepsin B, human cathepsin L, and cryptopain-1, with K is in the picomolar range. Cryptostatin was active over a wide pH range and was highly stable under physiological conditions. The protein was thermostable and retained its inhibitory activity even after incubation at 95°C. Cryptostatin formed tight complexes with cysteine proteases, so the complexes remained intact in the presence of sodium dodecyl sulfate and β-mercaptoethanol, but they were disassembled by boiling. An immunogold electron microscopy analysis demonstrated diffused localization of cryptostatin within oocystes and meronts, but not within trophozoites, which suggests a possible role for cryptostatin in host cell invasion by C. parvum.

Cryptopain-1, a cysteine protease of Cryptosporidium parvum , does not require the pro-domain for folding

SUMMARY Cryptosporidium parvum is an intracellular protozoan parasite that causes cryptosporidiosis in mammals including humans. In the current study, the gene encoding the cysteine protease of C. parvum (cryptopain-1) was identified and the biochemical properties of the recombinant enzyme were characterized. Cryptopain-1 shared common structural properties with cathepsin L-like papain family enzymes, but lacked a typical signal peptide sequence and contained a possible transmembrane domain near the amino terminus and a unique insert in the front of the mature domain. The recombinant cryptopain-1 expressed in Escherichia coli and refolded to the active form showed typical biochemical properties of cathepsin L-like enzymes. The folding determinant of cryptopain-1 was characterized through multiple constructs with or without different lengths of the pro-domain of the enzyme expressed in E. coli and assessment of their refolding abilities. All constructs, except one that did not contain the full-length mature domain, successfully refolded into the active enzymes, suggesting that cryptopain-1 did not require the pro-domain for folding. Western blot analysis showed that cryptopain-1 was expressed in the sporozoites and the enzyme preferentially degraded proteins, including collagen and fibronectin, but not globular proteins. This suggested a probable role for cryptopain-1 in host cell invasion and/or egression by the parasite.