Characterization of the Ashbya gossypii secreted N-glycome and genomic insights into its N-glycosylation pathway
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- 作者：Tatiana Q. Aguiar ; Hannu Maaheimo ; Annamari Heiskanen ; Marilyn G. Wiebe ; Merja Penttil? ; Luc¨ªlia Domingues
- 关键词：CRM ; complex rich medium ; DMM ; defined minimal medium ; DQF-COSY ; double quantum filtered correlation spectroscopy ; ENGase ; endo-¦Â-N-acetylglucosaminidase ; ER ; endoplasmic reticulum ; F ; deoxyhexose ; GH85 ; glycoside hydrolase family 85 ; GH92 ; glycoside hyd
- 刊名：Carbohydrate Research
- 出版年：2013
- 出版时间：15 November, 2013
- 年：2013
- 卷：381
- 期：Complete
- 页码：19-27
- 全文大小：1075 K
文摘
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.