Non-covalently Bound Cell Wall Proteins

Non-covalently bound CWPs may be associated with the cell wall by electrostatic or van-der-Waals forces, linking them to the cell wall polysaccharides. Otherwise, such proteins could also be directed to the external milieu, being on their way through the

cell wall. Proteins found in the external milieu often have hydrolytic or oxidative func-tions acting either in providing nutrients, in self-defence or in host infection (Chaffin, 2008). While enzymes involved in nutrient supply differ significantly between fungal species depending on their environmental divergence and their natural habitat, en-zymes involved in the formation and rebuilding of the cell wall structure are conserved to a certain extend over all fungal species (Mouyna et al., 2000a,b).

By the measurement of direct cell-wall-linked catalytic activity in the cell wall (pure cell wall fraction or whole cells) several enzymes were detected [for an extensive review see Rast et al. (2003)]. Most of these enzymes appear to be involved in the synthesis and rebuilding of the cell wall (Rast et al., 2003), being characterized as glucanases, transglycosylases and chitinases (Adams, 2004). Such cell wall-synthesizing enzymes are up to now best studied in the bakers’ yeast S. cerevisiae [reviewed in Lesage &

Bussey (2006)] and in the human pathogen C. albicans (Chaffin, 2008). Fungal glucan synthases, responsible for the synthesis ofβ-1,3-glucan, were described for several fungal species from the phylum ascomycota such as e.g. Yarrowia lipolytica (Kellner et al., 2005), Aspergillus nidulans (Beauvais et al., 2001) and A. fumigatus (Ibrahim et al., 2005). Also enzymes involved in the synthesis of β-1,6-glucan and several chitinases were already examined in C. albicans and S. cerevisiae. For comprehensive reviews on the enzymes involved in the cell wall synthesis in these two yeasts see references Lesage & Bussey (2006) and Chaffin (2008). The cell wall synthesis of filamentous fungi is possibly best understood in the human pathogen A. fumigatus (Bernard & Latg´e, 2001). Though not as comprehensively studied as the yeast cell walls, many enzymes involved in the wall biogenesis were characterized at the molecular level. Proteins being homologues to the glucan synthase complex of S. cerevisiae and chitinases are known fromA. fumigatus. Also two proteins involved in the synthesis ofα-1,3-glucan, a polysaccharide specific for Aspergillus spp., were identified as putative hydrolases with synthase domains (Bernard & Latg´e, 2001).

An extensively studied family of cell wall remodeling enzymes is the Gas-protein-family (Arroyo et al., 2007). The Gas-protein Gas-protein-family is a Gas-protein-family of GPI-anchored β-1,3-glucanosyl-transferases involved in the cell wall biogenesis, acting as β-1,3-glucan processing enzymes (Arroyo et al., 2007; Hartland et al., 1991; Mouyna et al., 2000b).

Members of this family are extremely well conserved in S. cerevisiae, C. albicans and Aspergillus species (Arroyo et al., 2007). These enzymes catalyze the cleavage of an internal glycosidic linkage of a β-1,3-glucan chain, releasing a reducing end and finally

transferring it to a non-reducing end of another β-1,3-glucan (Hartland et al., 1991;

Mouyna et al., 2000b). Thus, the members of the Gas-protein family act similar to glycoside hydrolases. The Gas-proteins were grouped into glycoside hydrolase family 72 (GH72) in the carbohydrate active enzyme database (CAZy; http://www.cazy.org/).

Hardly anything is known about the CWPs of higher basidiomycetes (Agaricomy-cotina). However, many of the fungi from this subphylum are involved in degrada-tion of complex substrates such as wood and other lignocellulosic substrates and are responsible for the mineralization of wooden biomass and decomposing of organic ma-terials. For this purpose, many basidiomycetes produce numerous enzymes responsible for degradation of plant cell wall material, such as laccases, manganese-dependent (or -independent) peroxidases, cellulases, and xylanases (Bouws et al., 2008; Cohen et al., 2002; Conesa et al., 2002; Morozova et al., 2007; Ng, 2004). Certain individual enzymes have already been detected within the fungal cell wall or the associated hyphal sheath (Barrasa et al., 1998; Ruel & Joseleau, 1991). For example, Barrasa et al. (1998) showed the association of aryl-alcohol oxidase in Pleurotus eryngii with the cell wall glycans of the fungus during the degradation of wheat straw.

There is ample of evidence that also typically intracellular proteins are attached to the fungal cell wall, such as glycolytic enzymes and other high abundant cytosolic proteins (Chaffin et al., 1998; Delgado et al., 2003; Edwards et al., 1999; Motshwene et al., 2003; Urban et al., 2003). They are generally released by extraction of the pure cell wall fraction withβ-mercaptoethanol. In the ascomycetous yeastsS. cerevisiae and C. albicans, glycolytic enzymes and chaperones (proteins assisting unfolded proteins to fold correctly) were shown to be attached to the cell wall (Alloush et al., 1997;

Eroles et al., 1997; Gil-Navarro et al., 1997; L´opez-Ribot & Chaffin, 1996; L´opez-Ribot et al., 1996). Enzymes known to be involved in glycolytic processes in the intracellular space such as 3-phosphoglycerate kinase (Alloush et al., 1997) and glyceraldehyde-3-phosphate dehydrogenase (Gil-Navarro et al., 1997) are shown to be located in the cell wall ofC. albicans. Enolase, another enzyme involved in glycolysis, and several proteins of the heat shock protein family were also detected (Edwards et al., 1999; Eroles et al., 1997; Russo et al., 1992). All these enzymes lack the well described classical N-terminal secretion signal and it remains unknown how they reach the cell surface. However, it has been speculated that these proteins are transported to the cell surface by a non-conventional export pathway (De Groot et al., 2005). Another assumption claims that these proteins hitch-hick in small amounts to the cell surface by leaking into vesicles

during the formation of transport vesicles (De Groot et al., 2005). This theory would be in agreement with the fact that until now only high abundant proteins such as heath shock proteins and glycolytic enzymes were detected in the cell wall. However, another explanation for the occurrence of these typically intracellular proteins in the cell wall could be the following: proteins may indeed derive from aging cells or cells damaged by shear stress. The cell walls of fungi are mostly negatively charged because of large numbers of phosphate groups, forming phosphodiester bridges. Thus, it might be possible that normally intracellular proteins with a relatively high isoelectric point (IP) bind to the mostly negatively charged cell wall (De Groot et al., 2005). Nevertheless, it seems to be unlikely that those typically intracellular proteins are only contaminations since there is evidence fromC. albicans that they play a role during infection of the host which was proven by immunoblotting and indirect immunofluorescence detection (Eroles et al., 1997). Further, Pardo et al. (1999) showed that also regenerating protoplasts secrete glycolytic enzymes. Nombela et al. (2006) suggested that these unconventional cell wall proteins are so called moonlighting proteins, performing multiple functions depending on their location.