Approaching the Secrets of N-Glycosylation in Aspergillus fumigatus: Characterization of the AfOch1 Protein

Abstract

The mannosyltransferase Och1 is the key enzyme for synthesis of elaborated protein N-glycans in yeast. In filamentous fungi genes implicated in outer chain formation are present, but their function is unclear. In this study we have analyzed the Och1 protein of Aspergillus fumigatus. We provide first evidence that poly-mannosylated N-glycans exist in A. fumigatus and that their synthesis requires AfOch1 activity. This implies that AfOch1 plays a similar role as S. cerevisiae ScOch1 in the initiation of an N-glycan outer chain. A Δafoch1 mutant showed normal growth under standard and various stress conditions including elevated temperature, cell wall and oxidative stress. However, sporulation of this mutant was dramatically reduced in the presence of high calcium concentrations, suggesting that certain proteins engaged in sporulation require N-glycan outer chains to be fully functional. A characteristic feature of AfOch1 and Och1 homologues from other filamentous fungi is a signal peptide that clearly distinguishes them from their yeast counterparts. However, this difference does not appear to have consequences for its localization in the Golgi. Replacing the signal peptide of AfOch1 by a membrane anchor had no impact on its ability to complement the sporulation defect of the Δafoch1 strain. The mutant triggered a normal cytokine response in infected murine macrophages, arguing against a role of outer chains as relevant Aspergillus pathogen associated molecular patterns. Infection experiments provided no evidence for attenuation in virulence; in fact, according to our data the Δafoch1 mutant may even be slightly more virulent than the control strains.

Unraveling Unique Structure and Biosynthesis Pathway of N-Linked Glycans in Human Fungal Pathogen Cryptococcus neoformans by Glycomics Analysis

“…These results imply that the truncated outer chain structure in N-glycans might be tolerable for maintaining the cell wall integrity of C. neoformans, different from other yeast species such as S. cerevisiae and C. albicans with hypermannosylated outer chains (8). Similarly, it was also reported that the outer chain structure of N-glycans is not important for maintenance of cell wall integrity in the filamentous fungi A. fumigatus (25).…”

“…The Caoch1 null mutant with loss of the ␣1,6-linked polymannose backbone was shown be attenuated in virulence. However, similar infection experiments revealed no difference between the afoch1 null mutant and control strains, suggesting that N-glycan outer chains of A. fumigatus are not associated with virulence (25). To address whether the modification of N-glycan outer chains affects the virulence of C. neoformans, we tested the virulence of the Cnoch1⌬ mutant, which is defective in initiating extension of N-glycan outer chains, using a murine model of systemic cryptococcosis.…”

“…The OCH1 deletion mutants of other ascomycetous yeast species, such as Kluyveromyces lactis, Hansenula polymorpha, Yarrowia lipolytica, Schizosaccharomyces pombe, and Pichia pastoris, and an ascomycetous filamentous fungus Neurospora crassa also exhibit altered morphological phenotypes under stress conditions, suggesting that outer chain N-glycans are important for cell wall integrity in yeast and fungal species (19 -24). The only exception was observed in the ascomycetous filamentous fungus and opportunistic human pathogen Aspergillus fumigatus, where deletion of OCH1 does not affect normal growth even under various stress conditions (25).…”

Cryptococcus neoformans Dual GDP-Mannose Transporters and Their Role in Biology and Virulence

“…This is con- sistent with the findings of Park et al in studies of C. neoformans mutants unable to initiate Golgi ␣-1,6-mannose branching (3). In this regard, it is also notable that cryptococcal N-glycan extension is more limited than that of S. cerevisiae or C. albicans (2); this is also the case for A. fumigatus (54). Another possibility is that other C. neoformans nucleotide sugar transporters that are localized to the Golgi complex have sufficiently relaxed specificity to transport some amount of GDP-mannose.…”

Biosynthesis of the Fungal Cell Wall Polysaccharide Galactomannan Requires Intraluminal GDP-mannose

“…It is, however, difficult to ascribe the dramatic growth phenotype of the ⌬gmtA mutant to a specific carbohydrate, because the absence of the transporter has pleiotropic effects on glycosylation. The addition of mannose to N-glycans may occur in the Golgi apparatus of filamentous fungi, but this maturation step seems limited and dispensable for growth (49,50). Likewise, mannosylation of IPC is not required for growth (51).…”