Molecular Cloning of cDNA Encoding α-N-Acetylgalactosaminidase from Acremonium sp. and Its Expression in Yeast

Alpha-N-acetylgalactosaminidase (α-GalNAc-ase; EC 3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA, nagA, encoding α-GalNAc-ase from Acremonium sp. was cloned, sequenced, and expressed in yeast Saccharomyces cerevisiae. The nagA contains an open reading frame which encodes for 547 amino acid residues including 21 residues of a signal peptide in its N-terminal. The calculated molecular mass of mature protein from the deduced amino acid sequence of nagA is 57260 Da, which corresponds to the value obtained from SDS–PAGE of native and recombinant enzymes treated with endo-β-N-acetylglucosaminidase H. The amino acid sequence of NagA showed significant similarity to those of eukaryotic α-GalNAc-ases and α-galactosidases (α-Gal-ases), particularly α-Gal-ase A (AglA) from Aspergillus niger. Phylogenetic analysis revealed that NagA does not belong to the cluster of vertebrate α-GalNAc-ase and α-Gal-ase but forms another cluster with AglA and yeast α-Gal-ases. Thus, the evolutionary origin of the fungal α-GalNAc-ase is suggested to be different from that of vertebrate α-GalNAc-ase. This is the first report of a microbial α-GalNAc-ase gene.