Identification of Protein Spots

In total, 70 spots from all 2-DE gels analyzed in this study were positively identified by LC-MS²and the analysis revealed 31 different proteins. All except of seven proteins were already detected previously within the study of 3-days-oldC. cinerea cultures described in Chapter 3 (Tables 3.3 to 3.10). Amongst the seven newly detected proteins are three glycoside hydrolases (ID-numbers 141, 142, 143), three unknown proteins (ID-numbers

146, 147, 148) and a pyranose dehydrogenase number 144). Only 3 of these (ID-numbers 142, 144, 146) were positively identified in the fractionated secretome of day 3 of cultivation (Table 5.5). The detection of additional proteins compared to the 1-DE shotgun approach of samples with the same age (Chapter 3; Section 3.5.2) can be considered as biological variance which is with only 3 newly detected proteins on day 3 of cultivation very low. Four newly detected proteins appeared first at day 6 and continued to be present till day 12 of cultivation. It can not be fully excluded that the rather low number of newly detected proteins after day 3 of cultivation might be a result of the different techniques used for protein identification in this experimental setup and the 1-DE shotgun approach presented in Chapter 3 (Section 3.5.2). However, the much more sensitive 1-DE shotgun approach lead to the identification of also low abundant proteins due to the fact that protein concentration of the sample is about 30 times higher than for 2-DE analysis as performed in Chapter 3 [see Chapter 3; Section 3.5.2; Wolters et al. (2001)] and possibly 10 times higher as performed here with pooling of parallel spots from replicate 2-DE gels. Therefore it can be speculated that many more proteins were already present at day 3, in many cases only in low concentrations.

Such low abundant proteins are not detectable by a 2-DE approach and possibly had even a concentration under the detection limit of the RuBP-staining [about 0.5 ng protein; (Rabilloud et al., 2001)]. Nevertheless, the spot picking was optimized in this experimental setup in comparison to the 2-DE analysis presented in Chapter 3 (Section 3.4.1) due to pooling parallel spots. However, the 2-DE analysis is still far from a complete documentation of the secretome. With the shotgun approach (Chapter 3, Section 3.5.2) 99 more proteins were positively identified on day 3 of cultivation than with the 2-DE analysis presented here.

Overlapping of the identified spots between the examined days of cultivation ofC. cin-erea showed that the majority of proteins was detected on all of the examined days (Fig-ure 5.10). Only few proteins were found to be present on only two or three days of culti-vation (ID-numbers 2, 60, 137 on two days and ID-numbers 1, 4, 77, 136, 143, 147, 148 on three days) and even fewer were detected at only one day of cultivation (ID-numbers 8, 141, 146). The dynamics of the secretome of C. cinerea is thus not represented by a high variety of different proteins but rather reflects a change of concentration of single proteins. Figure 5.9 shows a selection of proteins changing significantly in their concentration during the time of cultivation (for an analysis of all positively identified spots see Tables A.8 and A.9 in the Appendix). At this place, a selection of proteins

changing in amounts during cultivation is discussed in further detail.

Several of the identified peptidases secreted by C. cinerea are changing significantly in their concentration during the time of cultivation (for representative examples see Figure 5.9 a, b and c; see also Tables A.8 and A.9, ID-numbers 6, 8, 11, 21, 23, 26, 39, 71, 77, 90 in the Appendix to Chapter 5), indicating that they are possibly specific in their functions and are possibly also responsible for different regulative events occurring in the extracellular space. For example, the concentration of serine peptidase SB/S8, present in the free secretome and the hyphal sheath (ID-number 6) showed a significant increase from day 3 to day 6 (Figure 5.9 c). In contrast, the concentration of the omnipresent metallopeptidase MA/M36 (ID-number 11; Figure 5.9 b) is almost stable over the time in the freely secreted and the hyphal sheath fraction but he concentration of the protein increases significantly from day 3 to day 6 in the cell wall fraction. Functionally, there is no obvious difference between serine and metallopeptidases (Rao et al., 1998) and there is no striking explanation for the compartmentation. Still, the pH value of the different compartments (free secretome and the hyphal sheath in contrast to the cell wall) might be one possible explanation for the compartmentation of the peptidases as they might have different pH optima for enzyme activity.

Generally, this study revealed a high number of peptidases represented by the most abundant spots of the free secretome and the hyphal sheath proteome. These findings confirm the results obtained previously in Chapter 3 (Section 3.5.2.3). As already discussed in Chapter 3, extracellular peptidases are known to provide nitrogen in form of amino acids by degradation of peptides and proteins in the substrate for microorganisms (Rao et al., 1998).

The change in the protein concentration of several peptidases (Figures 5.9 a to c) over the time of cultivation indicates a regulation of the peptidases possibly due to the de-crease of nitrogen in the medium during growth, which is known to be a regulation factor for the expression of peptidases (Rao et al., 1998). However, also regulative functions of extracellular peptidases in fungi were shown in literature. For example, different laccase isoforms of Pleurotus ostreatus were shown to be regulated positively and/or negatively by a peptidase from the culture supernatant (Palmieri et al., 2000, 2001).

Also, lignin peroxidase activity from P. chrysosporium showed a negative correlation with two specific peptidases in the culture supernatant (Dosoretz et al., 1990).

A similar time pattern as for the peptidases was observed for the six identified glycoside hydrolases (ID-numbers 1, 15, 17, 141, 142, 143; Table 5.5). For

exam-ple, glycoside hydrolase from family 5 (ID-number 17) and glycoside hydrolase from family 7 (ID-number 142) increase in their protein concentration from day 3 to day 9 in the free secretome and the extractable cell wall fraction and from day 3 to day 12 of cultivation in the free secretome and the hyphal sheath, respectively (Figures 5.9 f and e). Both glycoside hydrolases families are known to contain different en-doglycosidases, amongst others 1,3-β-glucosidase, known to hydrolyze 1,3-β-glucans (http://www.cazy.org/fam/acc fam.html). InC. cinerea, they might be involved in the degradation of the glucans forming the hyphal sheath or the restructuring of the cell wall as it was shown previously for other fungal species (e.g. Aspergillus species and S. cerevisiae) (Bowman & Free, 2006).

In addition, several putative oxidoreductases were detected amongst the freely se-creted and the hyphal sheath proteins. Copper radical oxidase (ID-number 4), similar to glyoxal oxidases, catalyzing the oxidization of aldehydes to carboxylic acids, and most likely involved in the supply of H₂O₂ (Whittaker et al., 1996) occurs in three different isoforms in the free secretome and the hyphal sheath. Two of the three putative isoforms disappeared over the time of cultivation, indicating either a selective degradation or a selective secretion of a single isoform. For interest, a similar occurrence of degradation of specific isoforms was shown for lignin peroxidase inP. chrysosporium (Glumoff et al., 1990). However, the one long-lasting isoform of the copper radical oxidase found here over the time in the C. cinerea secretome is not identical with the isoform shown to be phosphorylated on sugar chains originating from the N-glycosylation of the protein (Chapter 4; Section 4.4.2).

Pyranose dehydrogenase (Pyranose dehydrogenases (PDHs)), a sugar oxidoreductase from the family of pyranose oxidases (Volc et al., 2000) is oxidizing aldo-pyranoses (e.g. glucose) to 2-aldo-ketoses by using different kinds of benzoquinones as electron acceptor (Peterbauer & Volc, 2010; Volc et al., 2000). In C. cinerea, the pyranose dehydrogenase (ID-number 144) was present on all examined days of cultivation in the free secretome but only appeared on day 6 of cultivation in the hyphal sheath and continued to be present till day 12 of cultivation. PDH is stated to be an enzyme specific for litter decomposing fungi such as Agaricus bisporus (Sygmund et al., 2008).

In this work is proposed that PDH is involved in the breakdown of lignocellulose by litter decomposing fungi such as A. bisporus as the enzyme is catalytically related to fungal pyranose oxidase and cellobiose dehydrogenase (Peterbauer & Volc, 2010). Also in C. cinerea, PDH might play a similar role as in A. bisporus in the degradation of

lignocellulose contained in horse dung, the natural substrate ofC. cinerea (K¨ues, 2000).

This, however, explains not the occurrence of PDH in the artificial YMG medium used for the cultivation of the fungus in this experimental setup and a function for this enzyme is currently not visible.

Two putative FAD/FMN-containing oxidoreductases (ID-numbers 31 and 36) ap-peared in a constant amount during the whole time of growth in the free secretome and the hyphal sheath. This might be due to constant secretion over the whole growth period or due to extreme stability of the enzyme and resistance against degradation.

FAD/FMN-containing oxidoreductases are known to provide H₂O₂ for peroxidases as already stated in Chapter 3 (Section 3.5.2.1). However, as they might use sugars as substrate they might as well be involved in the supply of C-sources.