Proteome analysis of Monascus pilosus mutant CE50 with high monacolin K production by two-dimensional electrophoresis
Mingyong Zhang, Tsuyoshi Miyake
Industrial Technology Center of Okayama Prefecture
5301 Haga, Okayama 701-1296 Japan
Abstract: M. pilosus is used to brew beni-koji in Japan, and also could produce various polyketides including monacolin K. A mutant CE50 with high production of monacolin K and red pigments has selected from M.pilosus IFO 4520 using mutagen treatment. The growth substrates and optimal conditions have been clarified to enhance the production of monacolin K of the mutant. However, the intracellular factors are still not known. Two-dimensional electrophoresis (2-DE) was used to investigate specific changes in the proteome of M. pilosus mutant CE50 and the parental IFO4520. Because the fungi of CE50 contain a lot of red pigments and other interfering compounds at the growth stage of sporulation, four protein preparation protocols were compared as following: protocol 1, directly extract in 2-D lysis buffer; protocol 2, directly extract using phenol; protocol 3, firstly remove the red pigments and other contaminants with acetone and 10% TCA in water, and then extract in 2-D lysis buffer; and protocol 4, firstly remove the red pigments with acetone and then extracted using phenol. For wild type IFO4520, the proteins prepared by three protocols except the protocol 1 were suitable for 2-DE; but for the mutant CE50, only the protein prepared by protocol 3 is suitable for 2-DE. The proteins of CE50 from phenol extraction protocols (2 and 4) were very difficult to dissolve again in 2-D lysis buffer at the final step of protein preparation process. About 600 protein spots of M.pilosus at growth stage of sporulation could be determined in the 13cm pH 3-10NL 2-D gel, and about 40% protein spots showed different in the expression levels between IFO4520 and CE50, the correlation coefficient of protein profiles is about 0.651 using analysis of PDQuest software.
Keywords: Filamentous fungi, protein extraction, proteome, two-dimensional electrophoresis
Coronary heart disease is one of the major causes of death in the developed countries. Coronary heart disease actually is a wide assortment of diseases, but the basic manifestation of many of them is atherosclerosis, caused when fatty deposits called plaque build up on the inner walls of arteries. Cholesterol is a major component of the atherosclerotic plaque. Many scientists believe that a high level of cholesterol in the blood is a major contributor to the development of atherosclerosis. Since in humans the greater part of the cholesterol in the body is synthesized de novo, mostly in the liver, the search for drugs to inhibit cholesterol biosynthesis has long been pursued as a means to lower the level of plasma cholesterol and so help to prevent and treat atherosclerosis (Endo 1985a).
M. pilosus is used in brewing beni-koji in Japan, and able to produce various polyketides potentially like other moulds and it is revealed that one of them is monacolin K (Endo 1985b). Monacolin K is one of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which are frequently utilized in the treatment of hypercholesterolemia, and is one of the second metabolites, which associated with sporulation processes of fungi including in activation of sporulation, formation of pigments required for sporulation structures, production of toxic metabolites secreted by growing colonies at the approximate time of sporulation (Calvo et al 2002).
A lot of researches on Monascus focused on growth substrates and on optimal conditions applied to different types of fermentation systems to enhance the production of different useful metabolites, and decrease the production of the harmful products (Lakrod et al. 2003). It is known that the secondary metabolites produced during fermentation depends on several factors including the Monascus strain used, the composition of fermentation medium, the culture methods, oxygen availability (aeration), extractive fermentation or the co-culture with other fungi (Ju et al. 1999) etc. Nearly all of these studies are involved with the environment factors, little studies are about the regulation inside of the fungal cells.
A mutant CE50 has been selected from M. pilosus IFO4520 after treated by mutagen MNNG; this mutant has ten fold higher monacolin K production than the wild type and the ferment condition for high monacolin K production has also been clarified by Miyake et al (2003). But the intracellular regulation of monacolin K production of the mutant CE50 is not known. This present paper is a primary result about the differential expression profile of protein between M.pilosus wild type IFO4520 and its mutant CE50 using 2-dimentional electrophoresis.
2 Materials and methods
2.1Strains and growth conditions
Both wild-type (IFO 4520) and the mutant CE50 of M. pilosus were firstly inoculated on PDA plate at 30oC for 15days to form the spores. Spores were harvested from the plate with sterile 0.01% Tween-20 and filtered with sterile glass wool to remove the mycelia. The harvested spores were centrifuged at 6000 rpm for 5 min and washed once with sterile water. The washed spores were inoculated in 100ml liquid PD media and shake at 120 rpm at 25oC or 30oC till the red pigment appearance in the media of CE50. The inoculation usually takes about 96h. Mycelia and spores were collected using filtering through a glass filter, and stored at -80oC for use.
Fungi were ground in liquid N2 using a mortar and pestle. The powder was divided four parts and weighted, and then quickly freezed in liquid N2 and stored at -80oC for further processing.
Four protocols of protein preparation were compared:
Protocol 1 directly extract in 2-D lysis buffer
About 0.2 g ground sample powder was resuspended in 0.5 ml of the 2-D lyses buffer (8 M urea, 4% CHAPS, 60 mM DTT). The mixture was sonicated in ice-water bath for 20 min, and centrifuged at 13000 x g twice for 10min (20oC). The supernatant was used to 1-D or 2-D analysis.
Protocol 2 directly extract using phenol
Phenol extraction of proteins is based on the protocol for plant described by Wang et al (2003). About 0.2g ground powder was resuspended in 1.0ml phenol (Tris buffered, pH8.0) and 1.0ml extraction buffer (0.1M Tris-HCl pH8.8, 2%SDS, 100mM DTT and 0.9M glucose). The mixture was vortexed thoroughly for 5min and the phenol phase was separated by centrifugation at 13000 x g for 10min. The upper phenol phase was pipetted to a new tube and centrifuged again. Transfer and measure the phenol phase to a new tube and add five volume of ice-cold 0.1 M ammonium acetate in methanol, the mixture was stored at -20oC for at least 30min. Precipitated proteins were recovered at 13000 x g for 5min, and washed with cold 80% acetone four time. The final pellet was dried and dissolved in rehydration solution same with protocol 1, soinicated for 20 min and centrifuged at 13000 x g for 10min, the un-dissolved pellet was discarded.
Protocol 3 firstly remove the red pigments with acetone and then extract in 2-D lysis buffer
About 0.2g ground sample powder was resuspended in 2ml cold acetone. After vortexing thoroughly for 5min, the tubes were centrifuged at 13000 x g for 5 min at 4oC. The pellet was further suspended in 10% aqueous TCA and vortexed. After centrifuged at 13000 x g for 5min at 4oC, the resultant pellet was washed four times more with cold acetone till the acetone was colorless. Each time the pellet was resuspended completely by vortexing. The final pellet was dried at room temperature and used to protein extraction. The following processes are same with protocol 1.
Protocol 4 firstly remove the red pigments with acetone and then extracted using phenol
The ground sample was firstly extracted by acetone same with protocol 3. The acetone extracted powder was used to isolate protein by phenol same with protocol 2.
Protein was quantified with 2-D Quant Kit (Amersham) using bovine serum albumin as standard. This protein quantification is compatible with 2-D sample preparation reagents, such as 2%SDS, 1%DTT, 8M urea, 4%CHAPS and 2% pharmalyte and 2% IPG Buffer.
2.3.1 1-D electrophoresis
1-D electrophoresis was carried out using pre-cast SDS polyacrylamide (10%) gels (BioCRAFT). Samples were prepared in Laemmli buffer with 2-mercaptoethanol. After electrophoresis, proteins were visualized with 0.025% Coomassie brilliant blue R250 as staining protocols of Hoefer.
2.3.2 2-D electrophoresis
Rehydration of Immobiline Dry Strips (Amersham) was carried out employing an Immobiline Dry Strip Re-swelling Tray (Amersham) following manufactures instructions. IPG strips (pH 3–10 NL, 13 cm long) were used for the present study. Sample was centrifuged at 13000 x g for 10 min and insoluble fraction was discarded. The Immobiline dry strips were allowed to rehydrate with the samples in 8 M urea, 2%w/v CHAPS, 0.5%IPG buffer (Amersham), traces of bromophenol blue and 60mM DTT of rehydration solution at 20oC for 16 h. Final sample load per strip was approximately 80mg. The rehydrated strips were then subjected to IEF. IEF was performed using Ettan IPGphor isoelectric Focusing system (Amersham) at 20oC. Briefly, the strips were focused at 500 V for 1 h, 1000 V for 1 h and 8000 V for 4 h, with a total of 33 kVh accumulated. Prior to the second-dimensional SDS-PAGE, IPG strips were equilibrated for 15 min in equilibration solution containing 50mM Tris-HCl, pH8.8, 6M urea, 30%w/v glycerol, 2%w/v SDS and traces of bromophenol blue with 100mM DTT. A second equilibration was carried out for 15 min by adding iodoacetamide (250 mg/10 mL) instead of DTT in equilibration solution; 5 mL of equilibration solution was used. Second- dimensional vertical SDS-PAGE was performed using precast gels (12.5% Tris-HCl), and gradient gels (5–20% Tris-HCl), all 1 mm in thickness (BioCRAFT). Briefly, electrophoresis was performed until the bromophenol band had exited the gel. Gels were stained with 0.025% Coomassie brilliant blue R250 or the MS compatible silver staining Kit (Invitrogen).
The 1-DE and 2-DE images were processed using Quantity One and PDQuest software (Bio-Rad), respectively.
3 Results and discussion
3.1 M. pilosus mutant CE50 with high monacolin K production
The mutant CE50 was selected from the M. pilosus IFO4520 treated by N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and has a maximum monacolin K production activity, which was ten fold higher than the parental strain (IFO4520) (Miyake et al 2003). The CE50 is also able to produce red pigment remarkably and form apparently different giant colony from parental strain. The optimal condition for monacolin K production of CE50 is grown at 25oC in GSP medium containing soybean or PD medium.
3.2 Effects of various protein preparation processes for 2-DE
Filamentous fungi have an exceptionally strong cell wall (Nandakumar and Marten 2002), and thus in sample preparation for 2-DE, cell lysis is often the most difficult step. Therefore, the vigorous lysis method to break the old mycelia and spores was chosen by grinding in liquid N2. For grinding in liquid N2 to extract protein for 2-DE, at least three advantages are: (1) shorten time comparing with treatment with lysis enzyme, (2) does not introduce other proteins into the 2-DE gels, (3) inhibit the protease in the lower temperature during the lysis process. From the protein yield (Fig. 1), grinding in liquid N2 is an effective lysis method to break the mycelia and the spores of old fungi.
Fig.1 Protein yields from different protein preparation protocols. W2-4 and M2-4 were proteins isolated from IFO4520 (W) and CE50 (M) by the protocol 2-4 as described in text, respectively.
Especially at the growth stage of sporulation, mycelia and spores contain a lot of second metabolites including the red pigments in M.pilosus mutant CE50, which badly interfered quantification of protein (data not shown) and the results of 1-D and 2-DE (Fig.2, 3). The protein prepared by phenol extraction protocols (protocol 2 and 4) from CE 50 were very difficult to dissolve in 2-D rehydration buffer again in the final step of protein preparation process, therefore, protein yield was very low in these two protocols (Fig.1 M3 and M4).
For wild type fungi IFO4520, the proteins prepared by three protocols except the protocol 1: directly extraction with 2-D lysis buffer, were suitable for 2-DE; but for the mutant CE50, only the protein from protocol 3 is suitable for 2-DE. The proteins of CE50 from phenol extraction protocol (2) and (4) were very difficult to re-dissolve again in 2-D lysis buffer. However, different protocols have different accumulation effects on proteins; these might disturb analysis of the protein profiles or proteomics. The basic proteins were abundant by protocol 3 (Fig.3, W3); the acidic proteins were abundant by protocol 2 and 4 of the phenol extraction (Fig.3, W2 and W4). These results suggested that same protein preparation protocol should be taken for comparison of protein profile using 2-DE.
Fig.2 1-D electrophoresis of proteins from M. pilosus IFO4520 and the mutant CE50 by different protein extraction protocols. M, protein marker; 1-4, the protein preparation protocols as described in method section. About 20ug proteins were loaded, run in 10% polyacrylamide gel and visualized with CBB.
Fig.3 2-D electrophoresis of proteins from M.pilosus IFO4520 (W) and CE50 (M) at growth stages of sporulation. 1-4 indicated that samples were prepared by various protocols as described in method section.
Nandakumar and Marten (2002) has compared several protocols for extraction of fungal proteins for 2-DE. The most suitable protocol described by Nandakumar and Marten (2002) was not suitable for 2-DE protein preparation from the fungi of M.pilosus mutant CE50 at the growth stage of sporulation (data not shown). Meanwhile, their protocols asked to use enzyme to treat the fungi or the solution; these treatments introduce some proteins into the 2-D gel, which would interfered the analyses for proteomics. Our protocols do not need to treat with any enzymes, the nucleic acid could not be found when staining with silver staining process.
3.3 Difference of protein profiles between the wild type and the mutant
The 2-DE protein profile showed many different expression proteins between the wild type M.pilosus IFO4520 and the mutant CE50 (Fig.2). About 600 protein spots could be determined in the 13cm pH 3-10NL 2-D gels of IFO4520 and CE50, and about 40% protein spots showed different in the expression levels, the correlation coefficient of protein profiles is about 0.651 between IFO4520 and CE50 at development stage of sporulation using analysis of PDQuest sofrware.
Dr. Mingyong Zhang thanks to JSPS for postdoc fellowship and the research grant.
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