Mass spectrometry-based proteomics of fungal wall glycoproteins

doi:10.1016/j.tim.2007.10.011

Trends in Microbiology

Volume 16, Issue 1, January 2008, Pages 20-26

https://doi.org/10.1016/j.tim.2007.10.011 Get rights and content

The manifold functions of fungal wall glycoproteins include maintenance of cell wall integrity, homotypic and heterotypic adhesion, biofilm formation, acquisition of iron and sterols, protein degradation and coping with oxidative stress. Transcriptome studies indicate that the expression levels of most cell wall glycoproteins can vary widely and are tightly controlled. However, owing to their complex and variable glycosylation, fungal wall glycoproteins are difficult to analyze using traditional proteomics approaches. Recent advances in mass spectrometry-based proteomics have enabled rapid and sensitive identification and quantitation of fungal wall glycoproteins; this will be particularly useful for studying the dynamics of the subproteome of fungal wall glycoproteins, and for the development of novel vaccines and diagnostic tools.

Section snippets

Fungal wall glycoproteins and cell wall organization

Many fungi have a layered cell wall. For example, the wall of the ascomycetous yeast Saccharomyces cerevisiae consists of a skeletal inner layer, which is surrounded by a fibrillar outer layer consisting of glycoproteins that emanate outwards (Figure 1). In S. cerevisiae, cell wall glycoproteins account for up to 10% of the total biomass per cell. Most cell wall glycoproteins in this organism are glycosylphosphatidylinositol-modified cell wall proteins (GPI–CWPs), which are covalently linked to

The physiological importance of fungal wall glycoproteins

There are several categories of CWPs. CWPs are either covalently linked to cell wall polysaccharides through a glycosidic linkage or an ester bond, or to other cell wall proteins through disulfide bonds. They can also be noncovalently associated with cell wall polysaccharides through, for example, a glycan-binding domain; they can also be ionically bound to the many negative charges due to, for example, phosphodiester groups present in the O- and N-linked carbohydrate side chains of cell wall

MS identification of fungal wall glycoproteins

The term ‘proteomics’, which denotes analysis of the entire protein complement expressed by a genome, was initially coined in the context of 2D gel electrophoresis (2-DE; see Glossary). In a typical experiment, proteins are released from the insoluble polysaccharide network, and then separated by 2-DE by making use of differences in their isoelectric point and mass. The proteins are subsequently proteolytically fragmented and the resulting peptides are analyzed with MS to reveal their

MS quantitation of fungal wall glycoproteins

The relative quantitation of biological samples using 2-DE is achieved by comparing the staining intensities of protein spots on the gel, whereas MS-based approaches use stable isotope labels that enable comparisons of corresponding peptide peak areas from different samples. Stable isotope labels can be incorporated at various stages of the experiment [38] (Figure 2). For example, they can be introduced by metabolic labeling with stable isotope-labeled amino acids [stable isotope labeling with

How profiling of fungal wall glycoproteins could contribute to vaccine development and diagnostics

Some promising applications of cell wall proteomic research are related to vaccine development, diagnostics, biofilm studies and cell surface engineering. For example, CWP-profiling studies might help to identify suitable vaccine candidates, as also shown for bacterial cell surface proteins 45, 46. LC/MS/MS provides information for selecting the best candidate proteins and the most promising protein regions. Such information has wide-reaching implications. (i) Protein abundance. Abundant CWPs

Concluding remarks and future perspectives

A key challenge for fungal wall biology is to investigate the role of fungal wall glycoproteins in infection and disease, in biofilm formation and in morphogenesis. It is equally important to study the dynamics of fungal wall glycoproteins in relation to environmental conditions in a reliable, sensitive and high-throughput way. Comparison between species will reveal how the subproteome of fungal wall glycoproteins of each species is adapted to its particular niche. Direct MS analysis of the

Acknowledgements

F.K. acknowledges the financial support by the EU (Framework VI: STREP FUNGWALL LSHB-CT-2004–511952). We thank Stanley Brul and Henk Dekker for useful discussions and support.

Glossary

2-DE: 2D electrophoretic separation of proteins based on differences in their isoelectric point and mass.
Endo-H: endo-β-N-acetylglucosaminidase H, an endoglycosidase that cleaves the β-1,4-linked di-N-acetylchitobiose core of the asparagine-linked N-chains, leaving one N-acetylglucosamine residue remaining on the asparagine residue. In combination with MS, the cleavage reaction can be used to map the N-glycosylation sites of the glycoprotein.
ICAT: Isotope-coded affinity tag. Reagent that specifically

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Trends in Microbiology

ReviewMass spectrometry-based proteomics of fungal wall glycoproteins

Section snippets

Fungal wall glycoproteins and cell wall organization

The physiological importance of fungal wall glycoproteins

MS identification of fungal wall glycoproteins

MS quantitation of fungal wall glycoproteins

How profiling of fungal wall glycoproteins could contribute to vaccine development and diagnostics

Concluding remarks and future perspectives

Acknowledgements

Glossary

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Review
Mass spectrometry-based proteomics of fungal wall glycoproteins