Abstract
The riboflavin producer Ashbya gossypii is a filamentous hemiascomycete, closely related to the yeast Saccharomyces cerevisiae, that has been used as a model organism to study fungal developmental biology. It has also been explored as a host for the expression of recombinant proteins. However, although N-glycosylation plays important roles in protein secretion, morphogenesis, and the development of multicellular organisms, the N-glycan structures synthesised by A. gossypii had not been elucidated. In this study, we report the first characterization of A. gossypii N-glycans and provide valuable insights into their biosynthetic pathway. By combined matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry profiling and nuclear magnetic resonance (NMR) spectroscopy we determined that the A. gossypii secreted N-glycome is characterized by high-mannose type structures in the range Man4-18GlcNAc2, mostly containing neutral core-type N-glycans with 8-10 mannoses. Cultivation in defined minimal media induced the production of acidic mannosylphosphorylated N-glycans, generally more elongated than the neutral N-glycans. Truncated neutral N-glycan structures similar to those found in other filamentous fungi (Man4-7GlcNAc2) were detected, suggesting the possible existence of trimming activity in A. gossypii. Homologs for all of the S. cerevisiae genes known to be involved in the endoplasmatic reticulum and Golgi N-glycan processing were found in the A. gossypii genome. However, processing of N-glycans by A. gossypii differs considerably from that by S. cerevisiae, allowing much shorter N-glycans. Genes for two putative N-glycan processing enzymes were identified, that did not have homologs in S. cerevisiae.
Original language | English |
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Pages (from-to) | 19-27 |
Journal | Carbohydrate Research |
Volume | 381 |
DOIs | |
Publication status | Published - 1 Jan 2013 |
MoE publication type | A1 Journal article-refereed |
Funding
We thank Fundação para a Ciência e a Tecnologia (FCT), Portugal, for financial support through the project AshByofactory (PTDC/EBB-EBI/101985/2008—FCOMP-01-0124-FEDER-009701) and MIT-Portugal Program (Ph.D. grant SFRH/BD/39112/2007 to Tatiana Q. Aguiar). We also thank Dr. Olli Aitio (University of Helsinki) for helpful assistance in the interpretation of the NMR data.
Keywords
- Ashbya gossypii
- N-Glycan structure
- N-Glycosylation
- Secreted glycoproteins