Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-alpha-D-mannosidase for N-glycan engineering in Pichia pastoris

Therapeutic glycoprotein production in the widely used expression host Pich ia pastoris is hampered by the differences in the protein-linked carbohydra te biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human-compatible N-glycans in th is organism is the conversion of the yeast-type high-mannose glycans to mam malian-type high-mannose and/or complex glycans. In this perspective, we ha ve co-expressed an endoplasmic reticulum-targeted Trichoderma reesei 1,2-al pha -D-mannosidase with two glycoproteins: influenza virus haemagglutinin a nd Trypanosoma cruzi trans-sialidase. Analysis of the N-glycans of the two purified proteins showed a > 85% decrease in the number of alpha -1,2-linke d mannose residues. Moreover, the human-type high-mannose oligosaccharide M an(5)GlcNAc(2) was the major N-glycan of the glyco-engineered trans-sialida se, indicating that N-glycan engineering can be effectively accomplished in P. pastoris. (C) 2001 Federation of European Biochemical Societies. Publis hed by Elsevier Science B.V. All rights reserved.