title: Molecular cloning and characterization of leucine aminopeptidase from Fasciola gigantica.
abstract: M17 leucine aminopeptidase (LAP) is one of a family of metalloexopeptidases, of which short peptide fragments are cleaved from the N-terminals. In this study, the full length of cDNA encoding Fasciola gigantica LAP (FgLAP) was cloned from adult parasites. The amino acid sequences of FgLAP showed a high degree of identity (98%) with that from Fasciola hepatica and a low degree of identities (11% and 9%) with those from cattle and human. Phylogenetic analysis revealed that the FgLAP was closely related and grouped with F. hepatica LAP (FhLAP). Northern analysis showed that FgLAP transcriptional products have 1800 base pairs. Analysis by RNA in situ hybridization indicated that LAP gene was expressed in the cecal epithelial cells of adult parasites. A polyclonal antibody to a recombinant FgLAP (rFgLAP) detected the native LAP protein in various developmental stages of the parasite. In a functional test, this rFgLAP displayed aminolytic activity using a fluorogenic Leu-MCA substrate, and was significantly inhibited by bestatin. Its maximum activity was at pH 8.0 and enhanced by Mn(2+) ions. Localization of LAP proteins by immunohistochemistry and immunofluorescence techniques indicated that the enzyme was distributed in the apical cytoplasm of cecal epithelial cells. Because of its important metabolic role and fairly exposed position, FgLAP is a potential drug target and a possible vaccine candidate against fasciolosis.
authors.affiliation: Department of Pathobiology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, Thailand. scsrr@mahidol.ac.th
authors.forename: Narin
authors.initials: N
authors.lastname: Changklungmoa
authors.forename: Kulathida
authors.initials: K
authors.lastname: Chaithirayanon
authors.forename: Pornanan
authors.initials: P
authors.lastname: Kueakhai
authors.forename: Krai
authors.initials: K
authors.lastname: Meemon
authors.forename: Suda
authors.initials: S
authors.lastname: Riengrojpitak
authors.forename: Prasert
authors.initials: P
authors.lastname: Sobhon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry; D018509:DNA, Helminth / Q000737:chemistry; D005209:Fasciola / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D005210:Fasciola hepatica / Q000145:classification / Q000201:enzymology / Q000235:genetics; D005260:Female; D006801:Humans; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D008195:Lymnaea / Q000469:parasitology; D008297:Male; D008647:Mesocricetus; D008969:Molecular Sequence Data; D010802:Phylogeny; D018510:RNA, Helminth / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D018509:DNA, Helminth; D018510:RNA, Helminth; D012333:RNA, Messenger; D007931:Leucyl Aminopeptidase