Glycobiology Advance Access published May 26, 2008

Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales, 2109, Australia.
Glycobiology (Impact Factor: 3.15). 09/2008; 18(8):626-37. DOI: 10.1093/glycob/cwn044
Source: PubMed


Glycosylation of proteins is important for protein stability, secretion, and localization. In this study, we have investigated the glycan synthesis pathways of 12 filamentous fungi including those of medical/agricultural/industrial importance for which genomes have been recently sequenced. We have adopted a systems biology approach to combine the results from comparative genomics techniques with high confidence information on the enzymes and fungal glycan structures, reported in the literature. From this, we have developed a composite representation of the glycan synthesis pathways in filamentous fungi (both N- and O-linked). The N-glycosylation pathway in the cytoplasm and endoplasmic reticulum was found to be highly conserved evolutionarily across all the filamentous fungi considered in the study. In the final stages of N-glycan synthesis in the Golgi, filamentous fungi follow the high mannose pathway as in Saccharomyces cerevisiae, but the level of glycan mannosylation is reduced. Highly specialized N-glycan structures with galactofuranose residues, phosphodiesters, and other insufficiently trimmed structures have also been identified in the filamentous fungi. O-Linked glycosylation in filamentous fungi was seen to be highly conserved with many mannosyltransferases that are similar to those in S. cerevisiae. However, highly variable and diverse O-linked glycans also exist. We have developed a web resource for presenting the compiled data with user-friendly query options, which can be accessed at This resource can assist attempts to remodel glycosylation of recombinant proteins expressed in filamentous fungal hosts.

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Available from: Helena Nevalainen, Oct 06, 2015
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    • "Putative N-glycosylation sites also exist in various effector proteins that have been identified in other plant pathogenic fungi, including Pep1 (Doehlemann et al., 2009; Hemetsberger et al., 2012) and Pit1 (Doehlemann et al., 2011) in U. maydis and Ecp6 in C. fulvum (de Jonge et al., 2010). As components of the yeast N-glycosylation pathway are conserved in filamentous fungi (Deshpande et al., 2008), the N-glycosylation of effector proteins may be widely deployed by plant fungal pathogens as a common mechanism to regulate their function in evading host innate immunity. Therefore, it will be important in the future to use Alg3 as a means of identifying further effectors that are glycosylated in order to help determine their precise effector functions. "
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    ABSTRACT: Plant pathogenic fungi deploy secreted effectors to suppress plant immunity responses. These effectors operate either in the apoplast or within host cells, so they are putatively glycosylated, but the posttranslational regulation of their activities has not been explored. In this study, the ASPARAGINE-LINKED GLYCOSYLATION3 (ALG3)-mediated N-glycosylation of the effector, Secreted LysM Protein1 (Slp1), was found to be essential for its activity in the rice blast fungus Magnaporthe oryzae. ALG3 encodes an α-1,3-mannosyltransferase for protein N-glycosylation. Deletion of ALG3 resulted in the arrest of secondary infection hyphae and a significant reduction in virulence. We observed that Δalg3 mutants induced massive production of reactive oxygen species in host cells, in a similar manner to Δslp1 mutants, which is a key factor responsible for arresting infection hyphae of the mutants. Slp1 sequesters chitin oligosaccharides to avoid their recognition by the rice (Oryza sativa) chitin elicitor binding protein CEBiP and the induction of innate immune responses, including reactive oxygen species production. We demonstrate that Slp1 has three N-glycosylation sites and that simultaneous Alg3-mediated N-glycosylation of each site is required to maintain protein stability and the chitin binding activity of Slp1, which are essential for its effector function. These results indicate that Alg3-mediated N-glycosylation of Slp1 is required to evade host innate immunity.
    The Plant Cell 03/2014; 26(3). DOI:10.1105/tpc.114.123588 · 9.34 Impact Factor
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    • "On the note that glycans have an effect on the activity of a protein , one of the problems standing in the way for filamentous fungi becoming effective producers of pharmaceutical proteins targeted for human consumption is the fungal oligo-mannose type glycosylation . While still of high-mannose type N -glycosylation patterns, filamentous fungi are far more conservative than yeast that has the tendency to hyperglycosylate proteins (Deshpande et al., 2008). However, they still lack the terminal sialic acid residues, characteristic of human glycosylation and important for defining the function of the glycan. "
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    ABSTRACT: Hosts used for the production of recombinant proteins are typically high-protein secreting mutant strains that have been selected for a specific purpose, such as efficient production of cellulose-degrading enzymes. Somewhat surprisingly, sequencing of the genomes of a series of mutant strains of the cellulolytic Trichoderma reesei, widely used as an expression host for recombinant gene products, has shed very little light on the nature of changes that boost high-level protein secretion. While it is generally agreed and shown that protein secretion in filamentous fungi occurs mainly through the hyphal tip, there is growing evidence that secretion of proteins also takes place in sub-apical regions. Attempts to increase correct folding and thereby the yields of heterologous proteins in fungal hosts by co-expression of cellular chaperones and foldases have resulted in variable success; underlying reasons have been explored mainly at the transcriptional level. The observed physiological changes in fungal strains experiencing increasing stress through protein overexpression under strong gene promoters also reflect the challenge the host organisms are experiencing. It is evident, that as with other eukaryotes, fungal endoplasmic reticulum is a highly dynamic structure. Considering the above, there is an emerging body of work exploring the use of weaker expression promoters to avoid undue stress. Filamentous fungi have been hailed as candidates for the production of pharmaceutically relevant proteins for therapeutic use. One of the biggest challenges in terms of fungally produced heterologous gene products is their mode of glycosylation; fungi lack the functionally important terminal sialylation of the glycans that occurs in mammalian cells. Finally, exploration of the metabolic pathways and fluxes together with the development of sophisticated fermentation protocols may result in new strategies to produce recombinant proteins in filamentous fungi.
    Frontiers in Microbiology 02/2014; 5:75. DOI:10.3389/fmicb.2014.00075 · 3.99 Impact Factor
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    • "Protein glycosylation pathways of 12 filamentous fungal species were investigated using a systems biology approach and developing a composite representation [15]. The N-glycosylation pathway in the cytoplasm and ER was evolutionarily conserved across the species studied, and highly specialized N-glycan structures with galactofuranose residues, phosphodiesters, and other insufficiently trimmed structures were identified. "
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    ABSTRACT: With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity.
    PLoS ONE 12/2013; 8(12):e84690. DOI:10.1371/journal.pone.0084690 · 3.23 Impact Factor
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