Cloning and characterization of a novel cathepsin B-like cysteine proteinase from Angiostrongylus cantonensis.
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.
Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou, 510080, People's Republic of China.
Mei
M
Cheng
Xiao
X
Yang
Zhuoya
Z
Li
Hualiang
H
He
Zhenyu
Z
Qu
Ai
A
He
Zhongdao
Z
Wu
Ximei
X
Zhan
D000595:Amino Acid Sequence; D017185:Angiostrongylus cantonensis / Q000201:enzymology* / Q000235:genetics*; D000818:Animals; D003001:Cloning, Molecular; D016000:Cluster Analysis; D057056:Cysteine Proteases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005260:Female; D020869:Gene Expression Profiling; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010802:Phylogeny; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D011506:Proteins; D057056:Cysteine Proteases