N-glycan profiling of Aspergillus nidulans using solid-phase glycan extraction and mass spectrometry

Diana Anyaogu, Shuang Yang, Jakob B. Nielsen, Hui Zhang, Michael Betenbaugh, Uffe Hasbro Mortensen

Author address: 

Department of Systems Biology, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark

Abstract: 

Filamentous fungi from the Aspergillus species are widely used as cell factories for the production of chemicals and enzymes, especially Aspergillus niger and Aspergillus oryzae are used as protein producers. Fungi have a high secretion capacity in comparison to other eukaryotic expression systems as algae, yeast and insect cells. The majority of the secreted proteins are glycosylated, thus glycosylation plays an important role in the secretory pathway. Glycosylation is also important in the production of therapeutic proteins as it is involved in protein stability, ligand binding, immunogenicity and serum half-life. Furthermore the efficacy of many therapeutic proteins depends on correct glycosylation. Thus, understanding the glycosylation will enable the directed glycoengineering in Aspergilli to improve protein production. In the present study the Solid-Phase Glycan Extraction (SPGE) method was used to isolate and purify N-glycans from the secretome and whole cell lysates from Aspergillus nidulans. The mass of the glycans was determined using a MALDITOF MS. In addition, A. nidulans strains with mutations in the glycosylation pathway were analyzed and compared to the reference strain. This study shows that some of the mutations had an effect on the N-glycan profile, which shifted the profile towards glycans with a lower mass. The method presented here is thus very efficient for extracting N-glycans and for quantifying the relative abundance of different N-glycans in the secretome and whole cell lysate .
2013

abstract No: 

N/A

Full conference title: 

27th Fungal Genetics Conference
    • Fungal Genetics Conference 27th (2013)