Engineering of glycan specific expression systems is being undertaken in numerous different expression systems, including fungal expression systems. N-glycosylation, the post-translational attachment of specific oligosaccharides to asparagine residues in newly formed proteins, has been shown to be important in modulating the biochemical and/or pharmacological properties of proteins and may affect half-life, immunogenicity, activity, and/or efficacy of glycoproteins. The fidelity of expressed proteins may thus be maintained by engineering expression systems to produce native N-glycans. The alpha-1,2-mannosidases are key in the development of N-glycans in both higher and lower eukaryotes. Understanding their biochemistry and substrate specificity has important applications in the development of humanized protein expression systems. Here we report an analysis and biochemical characterization of alpha-1,2-mannosidase enzymes from the filamentous fungus Aspergillus nidulans. Two Class 1 alpha-1,2-mannosidases from A. nidulans are shown to be capable of reducing the oligosaccharide Man(9)GlcNAc(2) to Man(5)GlcNAc(2), the precursor for further complex N-glycan structures-this indicates that these enzymes may have overlapping and possibly redundant activities. The alpha-1,2-mannosidase IC enzyme appeared to digest these oligosaccharides more readily than the alpha-1,2-mannosidases IB enzyme even though the alpha-1,2-mannosidase IB had a higher specific activity towards the synthetic substrate Man-alpha-1,2-Man-OCH3. This difference may be related to certain structural differences which limit the access of these intermediates within the binding pocket to the catalytic region of the enzyme. Manipulation of the expression of these components of the N-glycan processing pathway will be an important step in glyco-engineering this important fungal expression strain.

Although filamentous fungi are used extensively for protein expression, their use for the production of heterologous glycoproteins is constrained by the types of N-glycan structures produced by filamentous fungi as compared to those naturally found on the glycoproteins. Attempts are underway to engineer the N-glycan synthetic pathways in filamentous fungi in order to produce fungal expression strains which can produce heterologous glycoproteins carrying specific N-glycan structures. To fully realize this goal, a detailed understanding of the genetic components of this pathway in filamentous fungi is required. In this review, we discuss the characterization of the α-mannosidase gene family in filamentous fungi and its implications for the elucidation of the N-glycan synthetic pathway.

We describe the cloning and sequence characterization of three Class I α-1,2-mannosidase genes from the filamentous fungus Aspergillus nidulans. We used degenerate PCR primers to amplify a portion of the α-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 α-1,2-mannosidase IB gene and the 1759 nt coding region of the α-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 α-1,2-mannosidases. The deduced protein sequences were aligned with 11 published Class I α-1,2-mannosidases to determine sequence relationships. All three genes exhibited high similarity to other fungal α-1,2-mannosidases. The α-1,2-mannosidase IB exhibited very high similarity to the Aspergillus satoi and Penicillium citrinum α-1,2-mannosidases and likely represents an orthologue of these genes. Phylogenetic analysis suggests that the three A. nidulans Class I α-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.

A Class 2 alpha-mannosidase gene was cloned and sequenced from the filamentous fungus Aspergillus nidulans. A portion of the gene was amplified using degenerate oligonucleotide primers which were designed based on similarity between the Saccharomyces cerevisiae vacuolar and rat ER/cytosolic Class 2 protein sequences. The PCR amplification product was used to isolate the full length gene, and DNA sequencing revealed a 3383 bp coding region containing three introns. The predicted 1049 amino acid reading frame contained six potential N-glycosylation sites and encoded a protein of 118 kDa. The protein sequence did not appear to 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 indicated that the A.nidulans protein sequence was more highly similar to the rat ER/cytosolic (Rij = 0.33) and S.cerevisiae vacuolar alpha-mannosidases (Rij = 0.43) than the rat and yeast sequences were to each other (Rij = 0.29). These three enzymes were found to be distantly related to other Class 2 sequences, and compose a third subgroup of Class 2 alpha-mannosidases, as shown by ClustalW sequence alignment.