Cloning and characterization of a novel cathepsin B-like cysteine proteinase from Angiostrongylus cantonensis

Abstract

Cysteine protease plays a key role in host-parasite interactions. In this study, we identified a novel gene encoding a cathepsin B-like cysteine protease (AcCBL1) from the cDNA library of Angiostrongysus cantonensis fourth-stage larvae (L4) and characterized its biological role in the parasite. Sequence and phylogeny analysis showed that AcCBL1 is related to other cathepsin B family members with the conserved catalytic triad (Cys, His, Asn) and diagnostic occluding loop. In addition, the sequence contains a specific "hemoglobinase motif" and might have a hemoglobinase (Hb)-degrading function. The recombinant AcCBL1 (rAcCBL1) exhibited the protease activity by gelation SDS/PAGE assay; rAcCBL1 can cleave the fluorogenic substrate Z-Arg-Arg-AMC, and the optimum pH was 5.5. The enzyme can hydrolyse several host proteins including Hb and human IgG in acidic pH, but low levels of hydrolysis were observed in neutral pH. Reverse transcription polymerase chain reaction revealed that AcCBL1 expression was detected throughout various developmental stages, L3, L4, adult male and female worms. Western blotting analysis indicated that AcCBL1 was an excretory/secretory product of L4 in mature form of protease. Immunolocalization demonstrated that AcCBL1 was mainly localized in the intestine of L4. These results suggest that rAcCBL1 may play an important role in the parasite nutrition uptake.

Characterization of Spirometra erinaceieuropaei Plerocercoid Cysteine Protease and Potential Application for Serodiagnosis of Sparganosis

“…Cysteine protease is a candidate diagnostic antigen with good specificity for some helminth infections, including Angiostrongylus cantonensis [ 33 ], Necator americanus [ 39 ], Toxocara canis [ 15 ], Paragonimus westermani [ 14 ], Schistosoma japonicum [ 40 ], Clonorchis sinensis [ 41 ], Taenia solium [ 42 ], and S . erinaceieuropaei [ 17 ].…”

“…The enzymatic activity of rSeCP was assayed by substrate gel electrophoresis with a 12% SDS–PAGE gel copolymerized with 0.1% gelatin as the substrate, as previously reported [ 32 , 33 ].…”

Angiostrongylus cantonensis in China

“…There are many genes of A. cantonensis having been cloned and analyzed. They are cysteine protease inhibitor , matrix metalloproteinase (Sun et al 2012), cathepsin B-like cysteine proteinase (Cheng et al 2012), protein disulfide isomerases , novel gene encoding 16 kDa protein (Li et al 2012a), cathepsin B (Han et al 2011), galectin-10 (Liu et al 2013, etc. These work establish the foundation for researching the vaccine against A. cantonensis and searching drug target of angiostrongylsis.…”

Expression of cysteine proteinases and cystatins in parasites and use of cysteine proteinase inhibitors in parasitic diseases. Part I: Helminths

“…One year later, sequence analysis of the gene encoding the identified cathepsin showed that it contains a specific hemoglobinase motif to hydrolyze several host proteins including hemoglobin and human IgG. Using WB, cathepsin B was found in E/S from 4 th stage larvae, and the investigators suggested its role in parasite nutrition [177] . In the same year, Brazilian investigators immunoblot A. cantonensis E/S against pooled sera from patients with angiostrongyliasis and healthy controls.…”