The emerging field of glycobiology has had a significant impact on the production of recombinant proteins. The post-translational modification of proteins by the addition of sugar residues can significantly affect protein stability, conformation and functional activity. Glycosylation also plays an important role in cell-cell and intracellular protein targeting, These factors can have important effects on the commercial development of recombinant products, particularly in the health care and pharmaceutical industries. During the recombinant production of therapeutic proteins, the composition and types of oligosaccharides can be very sensitive to host cells and culture conditions. Heterogeneity in chain composition, chain structure and the utilization of alternate glycosylation sites is common during the recombinant production of therapeutic proteins and may, as a result, affect such factors as serum half-life, antigenicity, resistance to proteases and biological activity. To obtain stable and functional recombinant proteins, methods must be developed which yield glycoproteins having highly uniform carbohydrate moieties. This could be achieved by post-purification processing of recombinant proteins with enzymes which modify the carbohydrate moieties in a highly specific and predictable manner. Alternately the custom production of recombinant glycoproteins could also be achieved in vivo through the genetic alteration of the glycosylation pathway in the host expression system. This method, while more difficult to develop, would provide a more effective and lasting means to produce glycoproteins with specific glycosylation patterns. Our objective is to characterize the complete set of carbohydrate processing enzymes produced by the filamentous fungus Aspergillus in order to tailor the glycosylation pattern of recombinant therapeutic proteins produced in this fungus. The downstream products of such N-glycan remodeling research will include highly uniform glycoforms for pharmaceutical drugs. In this paper, we report the isolation and cloning of a novel cytosolic mannosidase from Aspergillus nidulans. Sequence analysis revealed a coding region of 3383bp containing three short introns. The deduced amino acid sequence of the A. nidulans mannosidase gene showed considerable homology to both the rat cytosolic/ER and yeast vacuolar mannosidases and, in common with these enzymes, did not contain a recognizable transmembrane domain. Phylogenetic analysis indicated that these cytosolic enzymes form a closely related group (Group III) which is distinct from both the mannosidase I enzymes (Group I) and the mannosidase II/ lysosomal mannosidase (Group II) enzymes.
Full conference title:
3rd EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 3rd (1996)