Characterization of the Ashbya gossypii secreted N-glycome and genomic insights into its N-glycosylation pathway.
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.
IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Tatiana Q
TQ
Aguiar
Hannu
H
Maaheimo
Annamari
A
Heiskanen
Marilyn G
MG
Wiebe
Merja
M
Penttilä
Lucília
L
Domingues
D055169:Eremothecium / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*
D011134:Polysaccharides
1D; 2D; Ashbya gossypii; CRM; DMM; DQF-COSY; ENGase; ER; F; GH85; GH92; GlcNAc; H; HSQC; MALDI-TOF; Man; N; N-Glycan structure; N-Glycosylation; N-acetyl; N-acetylglucosamine; N-acetylhexosamine; NAc; NMR; NOESY; P; PNGase F; Secreted glycoproteins; TOCSY; complex rich medium; defined minimal medium; deoxyhexose; double quantum filtered correlation spectroscopy; endo-β-N-acetylglucosaminidase; endoplasmic reticulum; glycoside hydrolase family 85; glycoside hydrolase family 92; heteronuclear single quantum coherence; hexose; m/z; mannose; mass-to-charge ratio; matrix-assisted laser desorption-ionization time-of-flight; nuclear Overhauser enhancement spectroscopy; nuclear magnetic resonance; one-dimensional; peptide:N-glycosidase F; phosphate; total correlation spectroscopy; two-dimensional; volumes of air per volume of liquid per minute; vvm