4.1 Abstract
4.4.1 Phosphorylated Proteins Occur in the Secretome of C. cinerea
In first experiments, C. cinerea monokaryon Okayama 7 was grown in shaking cultures for 3 and 6 days in standard YMG medium with 1 mCi33P per culture. The secretome of the experimental cultures was isolated and fractionated in freely secreted proteins of the culture supernatant, hyphal sheath proteins and extractable cell wall proteins (including non-covalently bound proteins and proteins bound with disulphide bridges). Proteins of the three different fractions were separated by 2-DE and radioactively labeled spots were detected by exposure of the gels to imager plates (Figures 4.2 to 4.4). In total, 91, 94, and 243 protein spots were visible on the 2-DE gels of the free secretome, the hyphal sheath and the extractable cell wall proteome, respectively. Less then 1% of these spots were specifically labeled with 33P. In contrast, on the 2-DE gels of the intracellular
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Figure 4.2: 2-DE of the freely secreted proteins fromCoprinopsis cinereagrown at 37◦C in standard YMG medium (supplemented with 1 mCi33P-containing phos-phate) on day 3 (A) and day 6 (C) of cultivation and corresponding autora-diographies (B) and (D), respectively. Proteins were separated on 18 cm Immobiline DryStrip pH 3-10 (Amersham Biosciences) in the first dimen-sion and on a 12% SDS-PAGE in the second dimendimen-sion. Gels were stained with RuBP. The imager plate for autoradiography was exposed to the gel for 3 days and subsequently scanned using a fluorescence reader FLA-5100 (Fujifilm, D¨usseldorf, Germany) with 50 µm resolution.
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Figure 4.3: 2-DE of the hyphal sheath proteins fromC. cinerea grown at 37◦C in stan-dard YMG medium (supplemented with 1 mCi 33P-containing phosphate) on day 3 (A) and day 6 (C) of cultivation and corresponding autoradiogra-phies (B) and (D), respectively. For further experimental details compare legend of Figure 4.2.
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Figure 4.4: 2-DE of the extractable cell wall proteins from C. cinerea grown at 37◦C in standard YMG medium (supplemented with 1 mCi33P-containing phos-phate) on day 3 (A) and day 6 (C) of cultivation and corresponding au-toradiographies (B) and (D), respectively. For further experimental details compare legend of Figure 4.2.
protein fraction about 700 spots were detected and more than 40% are labeled with33P (see Figure A.1 in the Appendix to Chapter 4). The resulting autoradiographies from the different fractions of the secretome showed weak signals of specific33P-labeled spots (ID-numbers 31, 36, 43, 46, 54) in all fractions of the 3-days- and 6-days-old cultures with a high background on the imager plates. The gels and autoradiographies revealed that in the freely secreted protein fraction and the hyphal sheath fraction one spot (ID-number 43) and two spots (ID-numbers 43 and 46), respectively, disappeared from day 3 to day 6 (compare Figures 4.2 and 4.3). The extractable cell wall fraction shows no difference in the number and position of the 33P-labeled spots on the analyzed days.
In the following experiments, only day 3 was further examined.
Determination of the overall phosphorus content revealed that the C. cinerea stan-dard growth medium YMG had a phosphorus concentration of 1.9 mM (3.37 mg/50 ml).
In order to obtain stronger and clearer signals upon radioactive labeling on the imager plates for future experiments, inorganic phosphate of the standard YMG medium was precipitated with CaCl2 prior to fungal inoculation. Due to this precipitation, the total phosphorus concentration was reduced to 1.0 mM (0.69 mg/50 ml) (see Table A.6 in the Appendix). 1 mCi33P-containing phosphate was added and the monokaryon Okayama 7 was cultivated in the phosphate-reduced medium for 3 days at 37◦C in shaking cultures.
The reduced phosphate concentration in the culture medium had a general influence on the morphology of the fungus. The fungal pellets were observed to be smaller and more compact compared to the fungal pellets in standard YMG medium. Also a dras-tic decrease of mucilagous material, representing most likely soluble polysaccharides, in the culture supernatant was observed for the cultures with reduced phosphate content.
33P-labeled extracellular proteins from 3-days-old phosphate reduced cultures were frac-tionated into the freely secreted proteins, the hyphal sheath proteins and the extractable cell wall proteins as in Chapter 2. Previously, polysaccharide material in supernatant samples were shown to hamper 2-DE of the free secretome (Fragner et al., 2009) and, in accordance, the resolution of the protein pattern on the 2-DE gels was drastically improved for the secretome of the phosphate-reduced cultures. Most importantly, pro-tein profiles were only little changed compared to the fractionated secretome of cultures grown in standard YMG medium. Also autoradiography of the 2-DE gels from the fractionated secretome of phosphate-reduced cultures showed 33P-labeled spots in the secretome, the hyphal sheath proteome and amongst the extractable cell wall proteins (Figures 4.5, 4.6 and 4.7). However, the signal intensity of 33P-labeled proteins was
drastically enhanced showing strong and clear signals of 33P-labeled spots.
In the autoradiography of the 2-DE gels of the free secretome, six spots with a rela-tively strong signal were detected with corresponding spots on the RuBP-stained 2-DE gel. In addition, three spots with rather weak signals were visible on the imager plate;
however, these signals had no corresponding protein spots stained with RuBP [Ruthe-nium(II) tris(bathophenanthroline disulfonate); Figures 4.5 to 4.7]. Autoradiography of the hyphal sheath fraction showed a similar protein profile with also six 33P-labeled protein spots and autoradiography of the extractable cell wall fraction revealed three
33P-labeled protein spots. Compared to the previous gels performed with the fraction-ated secretome of standard YMG cultures, two additional proteins (ID-numbers 4, 62) were detected to be33P-labeled on the autoradiography of the phosphate-reduced YMG medium.
The spots corresponding to the autoradiography signals were picked from the 2-DE gels from the free secretome, the hyphal sheath proteome and the extractable cell wall proteome of the phosphate-reduced YMG cultures and analyzed by LC-MS2 (Liquid chromatography coupled mass spectrometry). In total from all gels, seven different pro-teins were positively identified (Table 4.1). Two FAD/FMN-containing oxidoreductases (ID-numbers 31 and 36), a copper radical oxidase (ID-number 4), an unknown manno-protein (ID-number 46) and two different glycoside hydrolases from the families 37 and 72 (ID-numbers 62 and 43) were detected from the free secretome and the hyphal sheath proteome, respectively. All of them were already identified previously when analyzing the different fractions of the C. cinerea secretome (Chapter 3; Section 3.4). Of these, the glycoside hydrolase from family 72 and the CBM (carbohydrate binding module) -containing protein were detected in two different spots.
Previously, the copper radical oxidase was shown to be present in three different spots (Chapter 3; Section 3.4.1) of more or less the same protein amount. The same multiple spot pattern was detected in this study (compare Figures 3.3 (A) and 4.5 (A)). However, only one of the three spots was found to have an incorporated phosphate group. Further in the hyphal sheath fraction, a glycoside hydrolase of the family 37 (ID-number 62) was found. The unknown mannoprotein (ID-number 46) was detected on the imager plates of the free secretome and the hyphal sheath as well as in the extractable cell wall proteome. Besides this unknown mannoprotein, also an uncharacterized protein with a CBM (ID-number 54) was detected in two radioactively labeled spots in the cell wall proteome.
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Figure 4.5: 2-DE (A) and corresponding autoradiography (B) of the freely secreted pro-teins and the deglycosylated propro-teins of this fraction (C and D) from 3 days old shaking cultures of Coprinopsis cinerea grown at 37◦C in phosphate reduced YMG medium. Proteins were separated on 18 cm Immobiline DryStrip pH 3-10 (Amersham Biosciences) in the first dimension and on a 12% SDS-PAGE in the second dimension. Gels were stained with RuBP.
The imager plate for autoradiography was exposed to the gel for 3 days and subsequently scanned using a fluorescence reader FLA-5100 (Fujifilm, D¨usseldorf, Germany) with 50 µm resolution. Protein deglycosylation was performed with PNGaseF according to the manufacturers recommendations (Sigma-Aldrich; Seelze, Germany).
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Figure 4.6: 2-DE (A) and corresponding autoradiography (B) of the hyphal sheath pro-teins from 3-days-old cultures ofC. cinerea. For further experimental details compare legend of Figure 4.5.
Table 4.1: Identified spots from 2-DE gels of the fractionated secretome of C. cinerea grown in liquid YMG medium at 37◦C with a corresponding signal in au-toradiography.
ID Protein Proposed protein Fraction** Number of Labeled after
accession function* different deglycosylation
number spots
4 EAU83456 Copper radical oxidase S 1
-31 EAU80813 Oxidoreductase (2) S, HS 1 +
36 EAU82165 Oxidoreductase (3) S, HS 1 +
43 EAU90116 Glycoside hydrolase fam 72 (1) S, HS 2 +
46 EAU83394 Unknown mannoprotein (3) S, HS, CW 1 +/-***
54 EAU91343 CBM-containing protein (1) CW 2
-62 EAU86196 Glycoside hydrolase fam 37 HS 1 n.a.
* The proposed protein function is assigned after the protein analysis documented in Chapter 3 (Section 3.4.2.1). Numbers in brackets refer to the order in which these proteins were found, see Chapter 3 (Section 3.4.2.1)
** S: supernatant proteins; HS: hyphal sheath proteins; CW: cell wall proteins
*** Upon deglycosylation, this spot lost radioactive labeling only in the cell wall fraction, but not amongst the freely secreted proteins.
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Figure 4.7: 2-DE (A) and corresponding autoradiography (B) of the extractable cell wall proteins and the deglycosylated cell wall proteins (C and D) from 3-days-old cultures of C. cinerea. For further experimental details compare legend of Figure 4.5.