Article pubs.acs.org/jpr
Spatially Resolving the Secretome within the Mycelium of the Cell Factory Aspergillus niger Pauline Krijgsheld,† A. F. Maarten Altelaar,‡,§ Harm Post,‡,§ Jeffrey H. Ringrose,‡,§ Wally H. Müller,∥ Albert J. R. Heck,‡,§ and Han A. B. Wösten*,† †
Microbiology and Kluyver Centre for Genomics of Industrial Fermentation, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands ‡ Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands § Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands ∥ Biomolecular Imaging, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands S Supporting Information *
ABSTRACT: Aspergillus niger is an important cell factory for the industrial production of enzymes. These enzymes are released into the culture medium, from which they can be easily isolated. Here, we determined with stable isotope dimethyl labeling the secretome of five concentric zones of 7-day-old xylose-grown colonies of A. niger that had either or not been treated with cycloheximide. As expected, cycloheximide blocked secretion of proteins at the periphery of the colony. Unexpectedly, protein release was increased by cycloheximide in the intermediate and central zones of the mycelium when compared to nontreated colonies. Electron microscopy indicated that this is due to partial degradation of the cell wall. In total, 124 proteins were identified in cycloheximide-treated colonies, of which 19 secreted proteins had not been identified before. Within the pool of 124 proteins, 53 secreted proteins were absent in nontreated colonies, and additionally, 35 proteins were released ≥4-fold in the central and subperipheral zones of cycloheximide-treated colonies when compared to nontreated colonies. The composition of the secretome in each of the five concentric zones differed. This study thus describes spatial release of proteins in A. niger, which is instrumental in understanding how fungi degrade complex substrates in nature. KEYWORDS: fungus, Aspergillus niger, heterogeneity, secretion, cycloheximide, quantitative proteomics, dimethyl labeling, secretome
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heterogenic with respect to gene expression.7−11 Twenty-five percent of the expressed genes show a ≥ 2-fold change in expression when the periphery and the center of macrocolonies are compared.10 Differential gene expression in A. niger is accompanied by heterogeneity in growth and protein secretion.10,12,13 Two growth zones are distinguished in macrocolonies. The primary growth zone is located at the periphery of the colony, whereas the secondary growth zone is located in the colony center. Newly synthesized secreted proteins are released by these two growth zones.13 The majority of these proteins is retained in the cell wall, and is subsequently released into the growth medium.12 Thus, proteins released by the central zone of a macrocolony were for a large part produced during the primary growth phase at the colony periphery. The secretome of Aspergillus depends on the culture method and the composition of the medium.14−17 For instance, βglucosidase and α-amylase of Aspergillus oryzae are released in the substrate during solid state fermentation but are trapped in
INTRODUCTION The genus Aspergillus comprises species that are among the most abundant fungi in the world. They contribute to element recycling in nature by degrading dead organic material. As such, they can cause spoilage of food and feed. Aspergillus species are also pathogens of plants, animals, and humans, and they can affect the quality of the indoor environment.1 On the other hand, aspergilli are beneficial for mankind. They are used at large scale in the production of food, metabolites, and enzymes. For instance, Aspergillus niger is used in the industry for the production of organic acids2,3 and a wide variety of enzymes.4,5 The production capacity of this fungus is illustrated by strains that secrete more than 30 g L−1 glucoamylase.6 A. niger grows by means of hyphae that grow at their tips and that branch subapically. As a result, a network of interconnected hyphae is formed that is called a mycelium or a colony. The center and periphery of the colony represent the oldest and youngest parts of the mycelium, respectively. The size of the colony depends on the growth conditions. For instance, centimeter-scale macrocolonies are formed on agar medium, whereas (sub)-millimeter microcolonies are formed in a submerged liquid culture. Both colony types of A. niger are © 2012 American Chemical Society
Received: November 22, 2011 Published: March 26, 2012 2807
dx.doi.org/10.1021/pr201157b | J. Proteome Res. 2012, 11, 2807−2818
Journal of Proteome Research
Article
Figure 1. Sample preparation to determine the secretome of concentric zones of colonies of A. niger. Sandwiched colonies were grown for 7 days on agar medium and then transferred for 24 h to a ring plate. The medium in each concentric well was collected, concentrated with Amicon columns, and digested in solution with LysC and trypsin. Peptides from samples of zones 1 and 3 were labeled via dimethyl labeling with a light label, those from zones 2 and 4 with an intermediate label, and those from zones 3 and 5 with a heavy label. Labeled peptides from zones 1, 2, and 3 (A) and zones 3, 4, and 5 were pooled (B). As a control, samples of zone 3 with a light and heavy label were pooled (C). The pools were analyzed with nano LC-MS/MS. In a duplo experiment, labels were swapped using peptides from a biological duplicate. This resulted in a light labeled zone 3 and 5, an intermediate labeled zone 1 and 3, and a heavy labeled zone 2 and 4.
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the cell wall during submerged cultivation.14 The carbon source15 and the pH17 also have a profound effect on the secretome. So far, proteomic studies have focused on whole cultures. Here, we describe the quantitative analysis of the secretome of five concentric zones of 7-day-old xylose-grown colonies of A. niger. It is shown that the protein profiles are qualitatively similar but differ quantitatively. Colonies were treated with the protein synthesis inhibitor cycloheximide to distinguish between secreted proteins that had been recently produced and those that had been produced earlier and that are slowly released from the cell wall. Unexpectedly, a higher number and quantity of proteins was released into the medium upon cycloheximide treatment. Treatment with this compound may thus be used in industrial fermentations to improve the yield and composition of proteins released into the culture medium.
MATERIALS AND METHODS
Strain and Culture Conditions
A. niger N402 was grown at 30 °C using 25 mM xylose or 25 mM maltose as a carbon source. Static cultures were grown on minimal agar medium (MM)18 as sandwiched cultures.13 To this end, a perforated polycarbonate (PC) membrane (0.1 μm pores, 76 mm diameter; Profiltra, Almere, Netherlands) was placed on the agar medium, which was topped with a 0.45 mm layer of 1.25% agarose. A 1 mm plug of mycelium was positioned in the center of the agarose layer and covered with another 76 mm wide PC membrane. After 7 days of growth, colonies were transferred for 24 h to a ring plate.12 The five concentric wells in this plate were filled with liquid MM. Liquid cultures were grown in transformation medium (TM).19 After 16 h of growth at 250 rpm, the mycelium was harvested using a Büchner funnel and washed with 250 mL 0.85% NaCl (Merck, Darmstadt, Germany). Ten grams of wet weight mycelium was transferred to a 1 L Erlenmeyer flask containing 150 mL of MM. After growing for 6 or 24 h, 0.1 mg 2808
dx.doi.org/10.1021/pr201157b | J. Proteome Res. 2012, 11, 2807−2818
Journal of Proteome Research
Article
mL−1 cycloheximide (Sigma C7698, Zwijndrecht, Netherlands), 0.1 mg mL−1 hygromycin (Hygrogold, Invivogen Europe Cayla SAS, Toulouse, France), or 50 μg mL−1 phleomycin (Invivogen, Invivogen Europe Cayla SAS, Toulouse, France) were either or not added to the culture medium and incubation was prolonged for 66 and 48 h, respectively.
for 4 h. Samples were diluted 4-fold with MQ and treated with 0.1 μg of trypsin (V528A, Trypsin-Gold-Mass Spec grade, Porcine Sp. Act 15,000 u mg−1 Promega, Madison, USA) for 16 h at 37 °C. Samples were desalted using μElution plates (Oasis Waters, Eschborn, Germany). To this end, the wells were washed twice with 100% acetonitrile (Merck, Darmstadt, Germany) containing 10% formic acid (FA, Merck, Darmstadt, Germany). Formic acid was added to the sample to a final concentration of 10% and loaded in the wells of the μElution plates in aliquots of 150 μL. The Waters Extraction plate manifold was used for vacuum extraction of the samples through the wells (Waters S.A.S., En Yvelines Cedex, France). Wells were washed three times with 10% FA before eluting with 5% FA in 50% acetonitrile (Merck, Darmstadt, Germany). Samples were dried in vacuo and reconstituted in 20 μL of 100 mM triethylammonium bicarbonate (Sigma-Aldrich, Zweindrecht, The Netherlands) before dimethyl labeling. Stable isotope dimethyl labeling was performed as previously described in a total volume of 50 μL.21,22 Labeling efficiency was tested by analyzing an aliquot of the labeled samples on a regular LC MS/MS run and comparing overall peptide signal intensities. In all cases, the labeling efficiency was >98%.
Localization of Protein Synthesis and Protein Secretion
Protein synthesis and secretion were monitored as described.13 Sandwiched cultures were labeled for 4 h with 185 kBq 14Camino acids (Perkin-Elmer, NEC-445E amino acid mixture, L[14C(U)]-specific activity 1.94 GBq milliatom−1). A polyvinylidene difluoride (PVDF) membrane (Immobulon-P, Millipore, Bedford, USA) was placed between the sandwiched colony and the agar medium to immobilize secreted proteins. Label was placed on top of the colony after absorbing it to a piece of rice paper (Steicher & Schuell, Dassel, Germany) the size of the colony. Colonies were fixed with 4% formaldehyde in phosphate buffered saline (PBS) for 1 h at room temperature. Sandwiched colonies and PVDF membranes were washed 3 times 60 min with 1% casamino acids (Becton, Dickinson and company, Le-Pont-De-Claix, France). After drying overnight at room temperature, colonies and PVDF membranes were exposed to Kodak Biomax XAR film (Kodak Industry, France).
Nano LC-LTQ-Orbitrap-MS
Stable isotope dimethyl labeling was performed in a final volume of 50 μL (see above). The labels used for each of the samples are listed in Supporting Information Table 1. Two microliters of a sample was mixed with 2 μL of (an)other samples. The volume was adjusted to 10 μL, and the final concentration of formic acid was 10%. Four microliters of the mixture was used for MS. Samples were analyzed on an LTQOrbitrap (Thermo, Bremen, Germany) that was connected to an Agilent 1200 HPLC system. Samples were delivered to a trap column (ReproSil-Pur C18- AQ, 3 mm [Dr. Maisch GmbH, Ammerbuch, Germany]; 20 mm × 100 μm inner diameter, packed in house) at 5 μL min−1 in 100% solvent A (0.1 M acetic acid in water). Next, peptides were eluted from the trap column onto an analytical column (ReproSil-Pur C18AQ, 3 mm [Dr. Maisch GmbH, Ammerbuch, Germany]; 40 cm × 50-μm inner diameter, packed in-house) at 100 nL min−1 in a 120 min stepped gradient from 0 to 28% solvent B (0.1 M acetic acid in 8:2 v/v ACN/water) in 60 min and 28−50% solvent B in 25 min. The eluent was sprayed via distal coated emitter tips connected to the analytical column. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS. Full-scan MS spectra (from m/z 350 to 1500) were acquired in the Orbitrap with a resolution of 60,000 at m/z 400 after accumulation to a target value of 500,000 in the linear ion trap. The three most intense ions were selected for collision-induced fragmentation in the linear ion trap at a normalized collision energy of 35% after accumulation to a target value of 10,000.
Protein Detection on SDS-PAGE
Proteins in medium samples were precipitated with five volumes of acetone (Merck, Darmstadt, Germany) for 16 h at −20 °C. Samples were centrifuged at 10.000g for 15 min and dissolved in 2× SDS sample buffer (20% glycerol [LPS Benelux B.V., Rosmalen, The Netherlands], 4% SDS [J.T. Baker, Deventer, The Netherlands], 100 mM Tris-HCL pH 6.8 [Roche, Mannheim Germany], 0.01% bromophenol blue [Across Organics, Geel, Belgium], and 5% β-mercaptoethanol [Sigma-Aldrich, St. Louis, France]). Samples were analyzed on 12% SDS-PAA gels and stained with 0.1% Coomassie Brilliant Blue G250 (Sigma-Aldrich, Steinheim, Germany) in 25% methanol and 10% acetic acid (Merck, Darmstadt, Germany). Low Molecular Weight Marker (14,000−70,000 Da) (Sigma, St. Louis, France) was used as marker. Sample Preparation and Dimethyl Labeling for Mass Spectrometry
Medium samples were concentrated 10-fold with ultraconcentration columns (4 mL Amicon ultracentrifugal filter units, 10 kDa cutoff; Milipore, Amsterdam, Netherlands) (Figure 1). To this end, 500 μL of culture medium was transferred to the column and subjected to centrifugation for 30 min at 4000g in a swing out rotor. The concentrated samples were diluted twice with 2.5 mL of PBS (Sigma-Aldrich, Zweindrecht, The Netherlands) followed by centrifugation for 30 min at 4000g. Proteins in the concentrated sample (final volume of 50 μL) were subjected to in-solution-digestion.20 To this end, 80 μL of 8 M urea (Merck, Darmstadt, Germany) in 400 mM ammonium bicarbonate (Fluka, Steinheim, Germany) was added. Proteins were reduced for 30 min at 50 °C in the presence of 2 mM DTT (Sigma-Aldrich, Zweindrecht, The Netherlands) and subsequently alkylated for 30 min at room temperature by adding 4.5 mM iodoacetamide (Sigma-Aldrich, Zweindrecht, The Netherlands). Digestion was performed with 0.1 μg of Lys-C (lysyl endopeptidase 129−02541, 10 Au, Wako Pure Chemical Industry, Osaka Japan) by incubation at 37 °C
Protein Identification and Quantification
Raw files were searched with Thermo Proteome Discoverer 1.2.0.207 (Thermo Fischer Scientific) and a Mascot search engine (version 2.2.1) software platform (Matrix Science, London, U.K.) using the DSM protein database of A. niger CBS 513.88.23 The following settings of Mascot were used: trypsin with 2 missed cleavages, mass tolerance of fragment ions of 0.6 Da, and carbamidomethyl (C) as fixed modification. Oxidation (M), light dimethyl (K- and N-term), intermediate-dimethyl (K- and N- term), and heavy-dimethyl (K- and N-term) were set as variable modifications. The following settings of 2809
dx.doi.org/10.1021/pr201157b | J. Proteome Res. 2012, 11, 2807−2818
Journal of Proteome Research
Article
Proteome discoverer were used: peptide score filter: Mascot (ion score) > 20, maximum peptide rank = 1, peptide length: 7−35, and a peptide mass deviation 12 ppm, 8 ppm, and 10 ppm for untreated samples, cycloheximide treated samples, and cycloheximide treated−nontreated samples, respectively. Magellan storage files (.mgf) were loaded in Thermo Proteome Discoverer 1.2 simultaneously and heavy/light, medium/heavy, and medium/light Quan values were calculated. A technical control experiment was performed with zone 3 that had been labeled with two different labels (Figure 1). The 2log ratios for each protein should be 0. Indeed, this was the case for ≥90% of the proteins (Supporting Information Figure 1). Since proteins can be identified in one sample zone only, files were also loaded with the option replaced Missing Quan values, which allows quantification of “on/off” situations. Proteins found in both experiments with at least one peptide were included in the analysis. Protein release was considered differential when the average 2log ratio value was higher than 2 or lower than −2 and at least higher than 1 or lower than −1 in both biological replicates. To calculate the False Discovery Rate (FDR), spectra were searched against a reverse protein database. The spectra of the quantification of concentric zones in nontreated and cycloheximide treated colonies had an FDR of
