Characterization of the class I alpha-mannosidase gene family in the filamentous fungus Aspergillus nidulans.

We describe the cloning and sequence characterization of three Class I alpha-1,2-mannosidase genes from the filamentous fungus Aspergillus nidulans. We used degenerate PCR primers to amplify a portion of the alpha-1,2-mannosidase IA gene and used the PCR fragment to isolate the 2495 nt genomic gene plus several hundred bases of flanking region. Putative introns were confirmed by RT-PCR. Coding regions of the genomic sequence were used to identify two additional members of the gene family by BLAST search of the A. nidulans EST sequencing database. Specific PCR primers were designed to amplify portions of these genes which were used to isolate the genomic sequences. The 1619 nt coding region of the alpha-1,2-mannosidase IB gene and the 1759 nt coding region of the alpha-1,2-mannosidase IC gene, plus flanking regions, were fully sequenced. All three genes appeared to encode type-II transmembrane proteins that are typical of Class I alpha-1,2-mannosidases. The deduced protein sequences were aligned with 11 published Class I alpha-1, 2-mannosidases to determine sequence relationships. All three genes exhibited high similarity to other fungal alpha-1,2-mannosidases. The alpha-1,2-mannosidase IB exhibited very high similarity to the Aspergillus satoi and Penicillium citrinum alpha-1,2-mannosidases and likely represents an orthologue of these genes. Phylogenetic analysis suggests that the three A. nidulans Class I alpha-1, 2-mannosidases arose from duplication events that occurred after the divergence of fungi from animals and insects. This is the first report of the existence of multiple Class I mannosidases in a single fungal species.