Approaching the secrets of N-glycosylation in Aspergillus fumigatus: characterization of the AfOch1 protein.

Andrea Kotz,Johannes Wagener,Frank Ebel,Françoise H Routier,Jakob Engel,Jürgen Heesemann,Ilse Jacobsen,Bernd Echtenacher,Kirsten Nielsen

doi:10.1371/journal.pone.0015729

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.

Highlights

Eukaryotic proteins that enter the secretory pathway in the endoplasmatic reticulum (ER) are glycosylated by distinct sets of enzymes that catalyze either the formation of N- or O-linked glycans
PCR amplification from A. fumigatus chromosomal and cDNA using primers och1-59 and och1-39 resulted in two amplicons of different size
Sequencing of the PCR product derived from cDNA revealed an ORF that differed from the one predicted in the genome entry AFUA_5G08580. (The sequence of the full length mRNA has been submitted to the EMBL data base: Accession number FR667640.) The fact that the mRNA was amplified using a primer localized 173 bp downstream of the STOP codon indicates the presence of a longer untranslated 39-region, similar to the one predicted for the homologous A. nidulans gene AN4716.4-T

Summary

Introduction

Eukaryotic proteins that enter the secretory pathway in the endoplasmatic reticulum (ER) are glycosylated by distinct sets of enzymes that catalyze either the formation of N- or O-linked glycans. In yeasts N-glycans are characterized by their distinct and complex high-mannose structures [1]. The Och proteins of S. cerevisiae and C. albicans are a1,6mannosyltransferases that initiate a distinct branch in the N-glycan core thereby providing the platform for the subsequent formation of a large poly-mannosylated outer chain [2,3]. The C. albicans mutant showed an attenuated virulence in a murine model of infection [3] and triggered a reduced cytokine response in infected murine macrophages, a phenotype that was linked to recognition of outer chains by the mannose receptor [4]

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Results

Conclusion