MOLECULAR-CLONING, PRIMARY STRUCTURE, AND PROPERTIES OF A NEW GLYCOAMIDASE FROM THE FUNGUS ASPERGILLUS-TUBIGENSIS

A new glycoamidase, ptide-N-4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase (PNGase) At, was discovered in the eukaryote Aspergillus tubi gensis. The enzyme was purified to homogeneity, and the DNA sequence w as determined by cloning in Escherichia coli. Over 80% of the deduced amino acid sequence was verified independently by Edman analysis and/o r electrospray ionization-mass spectrometry of protease fragments of n ative PNGase At. This glycoamidase contains 12 potential asparagine-li nked glycosylation sites, of which at least 9 sites are occupied with typical high mannose oligosaccharides. PNGase At consists of two non-i dentical glycosylated subunits that are derived from a single polypept ide gene precursor, Evidence is presented suggesting that autocatalysi s is involved in subunit formation, PNGase At is an important new tool for analysis of asparagine-linked glycans; it can hydrolyze a broad r ange of glycopeptides, including those with core-linked alpha 1-->6 or alpha 1-->3 fucose, under conditions not favorable with existing glyc oamidases.