IDENTIFICATION AND ANALYSIS OF A CLASS 2 ALPHA-MANNOSIDASE FROM ASPERGILLUS-NIDULANS

A Class 2 alpha-mannosidase gene was cloned and sequenced from the fil amentous fungus Aspergillus nidulans. A portion of the gene was amplif ied using degenerate oligonucleotide primers which were designed based on similarity between the Saccharomyces cerevisiae vacuolar and rat E R/cytosolic Class 2 protein sequences, The PCR amplification product w as used to isolate the full length gene, and DNA sequencing revealed a 3383 bp coding region containing three introns. The predicted 1049 am ino acid reading frame contained six potential N-glycosylation sites a nd encoded a protein of 118 kDa, The protein sequence did not appear t o encode a typical fungal signal sequence or membrane spanning domain, Although the cellular location of the A.nidulans mannosidase was not determined, experimental evidence suggested that it was located within a subcellular organelle. The Matchbox sequence similarity matrix indi cated that the A.nidulans protein sequence was more highly similar to the rat ER/cytosolic (Rij = 0.33) and S.cerevisiae vacuolar alpha-mann osidases (Rij = 0.43) than tbe rat and yeast sequences were to each ot her (Rij = 0.29), These three enzymes were found to be distantly relat ed to other Class 2 sequences, and compose a third subgroup of Class 2 alpha-mannosidases, as shown by ClustalW sequence alignment.