Molecular cloning of cDNA encoding alpha-N-acetylgalactosaminidase from Acremonium sp and its expression in yeast

Alpha-N-acetylgalactosaminidase (alpha -GaLNAc-ase; EC 3.2.1.49) is an exog lycosidase specific for the hydrolysis of terminal alpha -linked N-acetylga lactosamine in various sugar chains. The cDNA, nagA, encoding alpha -GalNAc -ase from Acremonium sp. was cloned, sequenced, and expressed in yeast Sacc haromyces cerevisiae. The nagA contains an open reading frame which encodes for 547 amino acid residues including 21 residues of a signal peptide in i ts N-terminal. The calculated molecular mass of mature protein from the ded uced amino acid sequence of nagA is 57260 Da, which corresponds to the valu e obtained from SDS-PAGE of native and recombinant enzymes treated with end o-beta -N-acetyl-glucosaminidase ii. The amino acid sequence of NagA showed significant similarity to those of eukaryotic alpha -GalNAc-ases and alpha -galactosidases (alpha -Gal-ases), particularly alpha -Gal-ase A (AglA) fr om Aspergillus niger. Phylogenetic analysis revealed that NagA does not bel ong to the cluster of vertebrate alpha -GalNAc-ase and alpha -Gal-ase but f orms another cluster with AglA and yeast alpha -Gal-ases. Thus, the evoluti onary origin of the fungal alpha -GalNAc-ase is suggested to be different f rom that of vertebrate alpha -GalNAc-ase. This is the first report of a mic robial alpha -GalNAc-ase gene. (C) 2000 Academic Press.