Molecular characterization of an α-N-acetylgalactosaminidase from Clonorchis sinensis

Abstract

The alpha-N-acetylgalactosaminidase (alpha-NAGAL) is an exoglycosidase that selectively cleaves terminal alpha-linked N-acetylgalactosamines from a variety of sugar chains. A complementary DNA (cDNA) clone encoding a novel Clonorchis sinensis alpha-NAGAL (Cs-alpha-NAGAL) was identified in the expressed sequence tags database of the adult C. sinensis liver fluke. The complete coding sequence was 1,308 bp long and encoded a 436-residue protein. The selected glycosidase was manually curated as alpha-NAGAL (EC 3.2.1.49) based on a composite bioinformatics analysis including a search for orthologues, comparative structure modeling, and the generation of a phylogenetic tree. One orthologue of Cs-alpha-NAGAL was the Rattus norvegicus alpha-NAGAL (accession number: NP_001012120) that does not exist in C. sinensis. Cs-alpha-NAGAL belongs to the GH27 family and the GH-D clan. A phylogenetic analysis revealed that the GH27 family of Cs-alpha-NAGAL was distinct from GH31 and GH36 within the GH-D clan. The putative 3D structure of Cs-alpha-NAGAL was built using SWISS-MODEL with a Gallus gallus alpha-NAGAL template (PDB code 1ktb chain A); this model demonstrated the superimposition of a TIM barrel fold (alpha/beta) structure and substrate binding pocket. Cs-alpha-NAGAL transcripts were detected in the adult worm and egg cDNA libraries of C. sinensis but not in the metacercaria. Recombinant Cs-alpha-NAGAL (rCs-alpha-NAGAL) was expressed in Escherichia coli, and the purified rCs-alpha-NAGAL was recognized specifically by the C. sinensis-infected human sera. This is the first report of an alpha-NAGAL protein in the Trematode class, suggesting that it is a potential diagnostic or vaccine candidate with strong antigenicity.