title: Molecular cloning and characterization of a cathepsin B from Angiostrongylus cantonensis.
abstract: Cysteine proteases, a superfamily of hydrolytic enzymes, have numerous functions in parasites. Here, we reported the cloning and characterization of a cDNA encoding a cathepsin B (AcCPB) from Angiostrongylus cantonensis fourth-stage larvae cDNA library. The deduced amino acid sequence analysis indicated AcCPB is related to other cathepsin B family members with an overall conserved architecture. AcCPB is evolutionarily more close to other parasitic nematode cathepsin B than the ones from hosts, sharing 43-53% similarities to the homologues from other organisms. Real-time quantitative PCR analysis revealed that AcCPB was expressed significantly higher in the fourth-stage larvae (L4) and the fifth-stage larvae (L5) than that in the third-stage larvae (L3) and adult worms (Aw). Unexpectedly, AcCPB was expressed at a higher level in L4 and L5 derived from mice than the larvae at the same stages derived from rats. The protease activity of recombinant AcCPB (rAcCPB) expressed in Escherichia coli showed high thermostability and acidic pH optima. The role in ovalbumin digestion and enzyme activity of rAcCPB could be evidently inhibited by cystatin from A.cantonensis. Furthermore, we found rAcCPB increased the expression levels of CD40, MHC II, and CD80 on LPS-stimulated dendritic cells (DCs). In this study, we provided the first experimental evidence for the expression of cathepsin B in A.cantonensis. Besides its highly specific expression in the stages of L4 and L5 when the worms cause dysfunction of the blood-brain barrier of hosts, AcCPB displayed different expression profiles in non-permissive host- and permissive host-derived larval stages and was involved in the maturation of DCs, suggesting a potential role in the central nervous system invasion and the immunoregulation during parasite-host interactions.
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou 510080, China.
authors.forename: Yan-ping
authors.initials: YP
authors.lastname: Han
authors.forename: Zheng-yu
authors.initials: ZY
authors.lastname: Li
authors.forename: Bao-chuan
authors.initials: BC
authors.lastname: Li
authors.forename: Xi
authors.initials: X
authors.lastname: Sun
authors.forename: Cheng-cheng
authors.initials: CC
authors.lastname: Zhu
authors.forename: Xiao-ting
authors.initials: XT
authors.lastname: Ling
authors.forename: Huan-qin
authors.initials: HQ
authors.lastname: Zheng
authors.forename: Zhong-dao
authors.initials: ZD
authors.lastname: Wu
authors.forename: Zhi-yue
authors.initials: ZY
authors.lastname: Lv
mesh_terms: D000595:Amino Acid Sequence; D017185:Angiostrongylus cantonensis / Q000201:enzymology* / Q000235:genetics*; D000818:Animals; D002401:Cathepsin B / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D016000:Cluster Analysis; D003713:Dendritic Cells / Q000276:immunology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010802:Phylogeny; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D002401:Cathepsin B