Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica

Sansri, Veerawat; Changklungmoa, Narin; Chaichanasak, Pannigan; Sobhon, Prasert; Meemon, Krai

doi:10.1016/j.actatropica.2013.06.013

Cited by 26 publications

(17 citation statements)

References 32 publications

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“…Clades 1, 2, and 5 belonged to adultspecific cathepsin L, while clades 3 and 4 were classified as juvenile-specific cathepsin L (Robinson et al 2008;Sansri et al 2013;Cwiklinski et al 2015). Two juvenile cathepsin L proteases have been identified in F. hepatica, namely FhCL3 and FhCL4 (Harmsen et al 2004;Cancela et al 2008), and their sequences were identical to cathepsin L1G (FgCatL1G) and L1H (FgCatL1H) of F. gigantica, respectively (Cancela et al 2008;Sansri et al 2013). FhCL3 exhibited optimal activity and stability at neutral pH and could cleave collagen, but not immunoglobulin, suggesting its role in parasite migration through the liver (Table 1) (Corvo et al 2009;Robinson et al 2011).…”

Section: Cathepsin Lmentioning

confidence: 98%

“…They can be classified into five clades according to their sequence identities (Robinson et al 2008;Sansri et al 2013;Cwiklinski et al 2015). Clades 1, 2, and 5 belonged to adultspecific cathepsin L, while clades 3 and 4 were classified as juvenile-specific cathepsin L (Robinson et al 2008;Sansri et al 2013;Cwiklinski et al 2015). Two juvenile cathepsin L proteases have been identified in F. hepatica, namely FhCL3 and FhCL4 (Harmsen et al 2004;Cancela et al 2008), and their sequences were identical to cathepsin L1G (FgCatL1G) and L1H (FgCatL1H) of F. gigantica, respectively (Cancela et al 2008;Sansri et al 2013).…”

Section: Cathepsin Lmentioning

confidence: 99%

Section: Cathepsin Lmentioning

confidence: 99%

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Juvenile-specific cathepsin proteases in Fasciola spp.: their characteristics and vaccine efficacies

Meemon

Sobhon

2015

Parasitol Res

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Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is one of the most neglected tropical zoonotic diseases. One sustainable control strategy against these infections is the employment of vaccines that target proteins essential for parasites' invasion and nutrition acquiring processes. Cathepsin proteases are the most abundantly expressed proteins in Fasciola spp. that have been tested successfully as vaccines against fasciolosis in experimental as well as large animals because of their important roles in digestion of nutrients, invasion, and migration. Specifically, juvenile-specific cathepsin proteases are the more effective vaccines because they could block the invasion and migration of juvenile parasites whose immune evasion mechanism has not yet been fully developed. Moreover, because of high sequence similarity and identity of cathepsins from juveniles with those of adults, the vaccines can attack both the juvenile and adult stages. In this article, the characteristics and vaccine potentials of juvenile-specific cathepsins, i.e., cathepsins L and B, of Fasciola spp. were reviewed.

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Section: Cathepsin Lmentioning

confidence: 98%

Section: Cathepsin Lmentioning

confidence: 99%

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Juvenile-specific cathepsin proteases in Fasciola spp.: their characteristics and vaccine efficacies

Meemon

Sobhon

2015

Parasitol Res

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“…A single colony of Pichia pastoris GS115 containing proFgCatL1H-inserted pPICZαB vector (Sansri et al 2013) was grown in BMMY [1 % (w/v) yeast extract, 2 % (w/v) peptone, 100 mM potassium phosphate, pH 6.0, 1.34 % (w/v) yeast nitrogen base, 5-10 % (w/v) d-biotin, and 0.5 % (v/v) methanol], for 72 h at 28-30°C with shaking. To induce expression of recombinant proFgCatL1H (rproFgCatL1H), methanol was added to the culture to a final concentration of 0.5 %, every 24 h. The rproFgCatL1H was purified by using nickel-nitrilotriacetic acid (Ni-NTA) affinity column as previously described (Sansri et al 2013).…”

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

“…Cathepsin Ls (CatLs) are the most abundant enzymes secreted by F. hepatica (Smith et al 1993;Collins et al 2004;Dalton et al 2003) and predominantly released in excretory-secretory (ES) products (Morphew et al 2011;Jefferies et al 2001). Sansri et al (2013) reported that F. gigantica cathepsin L1H (FgCatL1H) is the isotype highly expressed in the early stages, including metacercariae and newly excysted juvenile (NEJ). Its molecular weight is approximately 47.6 and 38.3 kDa in glycosylated and deglycosylated forms, respectively.…”

Section: Introductionmentioning

confidence: 99%

Monoclonal antibody against recombinant Fasciola gigantica cathepsin L1H could detect juvenile and adult cathepsin Ls of Fasciola gigantica

et al. 2014

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Cathepsin Ls (CatLs), the major cysteine protease secreted by Fasciola spp., are important for parasite digestion and tissue invasion. Fasciola gigantica cathepsin L1H (FgCatL1H) is the isotype expressed in the early stages for migration and invasion. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1H (rFgCatL1H) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with recombinant proFgCatL1H (rproFgCatL1H). This MoAb is an immunoglobulin (Ig)G1 with κ light chain isotype. The MoAb reacted specifically with rproFgCatL1H, the native FgCatL1H at a molecular weight (MW) 38 to 48 kDa in the extract of whole body (WB) of metacercariae and newly excysted juvenile (NEJ) and cross-reacted with rFgCatL1 and native FgCatLs at MW 25 to 28 kDa in WB of 2- and 4-week-old juveniles, adult, and adult excretory-secretory (ES) fractions by immunoblotting and indirect ELISA. It did not cross-react with antigens in WB fractions from other parasites, including Gigantocotyle explanatum, Paramphistomum cervi, Gastrothylax crumenifer, Eurytrema pancreaticum, Setaria labiato-papillosa, and Fischoederius cobboldi. By immunolocalization, MoAb against rFgCatL1H reacted with the native protein in the gut of metacercariae and NEJ and also cross-reacted with CatL1 in 2- and 4-week-old juveniles and adult F. gigantica. Therefore, FgCatL1H and its MoAb may be used for immunodiagnosis of both early and late fasciolosis in ruminants and humans.

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Identification and expression of Fasciola gigantica thioredoxin

et al. 2014

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In the present study, a cDNA encoding Trx from F. gigantica (FgTrx) was cloned by polymerase chain reaction (PCR). The sequence of FgTrx, analyzed by BLAST, SignalP, and ClustralW programs, showed 315 bp of an open reading frame (ORF), 12 bp 5'UTR, 78 bp 3'UTR, and the putative FgTrx peptide comprising of 104 amino acids, with a molecular weight of 11.68 kDa, with the active site containing five amino acids (tryptophan, cysteine, glycine, proline, cysteine) with a conserved dithiol motif from the two cysteines, and pI 5.86. The peptide had no signal sequence; hence, it was not a secreted protein. The recombinant FgTrx was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTrx). The FgTrx protein expression, estimated by indirect ELISA using the rabbit anti-rFgTrx as probe, showed high levels in eggs, 2- and 4-week-old juveniles, and adult parasite. In a functional test, the rFgTrx exhibited specific activity that could be suppressed by an inhibitor (PX12). When tested by immunoblotting and immunohistochemistry, rabbit anti-rFgTrx reacted with natural FgTrx at a molecular weight of 11.68 kDa from eggs, metacercariae, NEJ, 2- and 4-week-old juveniles, and adult F. gigantica. The FgTrx protein was distributed at high levels in the tegument of 2- and 4-week-old juveniles, and the tegument, parenchyma, eggs, and reproductive organs of adult parasites. FgTrx may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or drug target.

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Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica

Cited by 26 publications

References 32 publications

Juvenile-specific cathepsin proteases in Fasciola spp.: their characteristics and vaccine efficacies

Juvenile-specific cathepsin proteases in Fasciola spp.: their characteristics and vaccine efficacies

Monoclonal antibody against recombinant Fasciola gigantica cathepsin L1H could detect juvenile and adult cathepsin Ls of Fasciola gigantica

Identification and expression of Fasciola gigantica thioredoxin

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