Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from Angiostrongylus cantonensis.
Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, a novel cystatin molecule (AcCystatin) was cloned from a cDNA library of Angiostrongylus cantonensis fourth-stage larvae. The putative 14-kDa protein contained 120 residues with cystatin-conserved motifs known to interact with the active site of cysteine peptidases and showed high identities with cystatins from other nematodes. RT-PCR analysis revealed that the expression pattern of AcCystatin was equal at the time points of third-stage larvae, fourth-stage larvae, and adults of the parasite life cycle. The recombinant AcCystatin (rAcCystatin) expressed and purified from Escherichia coli has been demonstrated to possess an obvious inhibitory activity against cathepsin B and could significantly upregulate nitric oxide production from IFN-gamma activated RAW 264.7 macrophages. Sera from mice (non-permissive host) infected with A. cantonensis detected rAcCystatin by Western blot, while the sera from infected rats (permissive host) could not. The results implied that AcCystatin might be an immunoregulator in A. cantonensis infection.
Liu
Yu-Hong
YH
Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou, 510080, China.
Han
Yan-Ping
YP
Li
Zheng-Yu
ZY
Wei
Jie
J
He
Han-Jiang
HJ
Xu
Chang-Zhi
CZ
Zheng
Huan-Qin
HQ
Zhan
Xi-Mei
XM
Wu
Zhong-Dao
ZD
Lv
Zhi-Yue
ZY
D020816:Amino Acid Motifs; D017185:Angiostrongylus cantonensis / Q000201:enzymology* / Q000235:genetics*; D000818:Animals; D002401:Cathepsin B / Q000037:antagonists & inhibitors; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D015891:Cystatins / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009569:Nitric Oxide / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D017206:Strongylida Infections / Q000469:parasitology
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D015801:Helminth Proteins; D011994:Recombinant Proteins; D009569:Nitric Oxide; D002401:Cathepsin B