Article pubs.acs.org/jpr
Proteome Alterations of Hippocampal Cells Caused by Clostridium botulinum C3 Exoenzyme Anke Schröder,* Astrid Rohrbeck, Ingo Just, and Andreas Pich Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany S Supporting Information *
ABSTRACT: C3bot from Clostridium botulinum is a bacterial mono-ADP-ribosylating enzyme, which transfers an ADP-ribose moiety onto the small GTPases Rho A/B/C. C3bot and the catalytic inactive mutant (C3E174Q) cause axonal and dendritic growth as well as branching in primary hippocampal neurons. In cultured murine hippocampal HT22 cells, protein abundances were analyzed in response to C3bot or C3E174Q treatment using a shotgun proteomics approach. Proteome analyses were performed at four time points over 6 days. More than 4000 protein groups were identified at each time point and quantified in triplicate analyses. On day one, 46 proteins showed an altered abundance, and after 6 days, more than 700 proteins responded to C3bot with an up- or down-regulation. In contrast, C3E174Q had no provable impact on protein abundance. Protein quantification was verified for several proteins by multiple reaction monitoring. Data analysis of altered proteins revealed different cellular processes that were affected by C3bot. They are particularly involved in mitochondrial and lysosomal processes, adhesion, carbohydrate and glucose metabolism, signal transduction, and nuclear proteins of translation and ribosome biogenesis. The results of this study gain novel insights into the function of C3bot in hippocampal cells. KEYWORDS: Clostridium botulinum, C3 exoenzyme, SILAC, shotgun proteomics
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INTRODUCTION Mono-ADP-ribosylation is a common catalytic activity of toxins expressed by pathogenic bacteria to alter functions of host target proteins. Binary ADP-ribosylating toxins are Clostridium botulinum C2 toxin, C. perf ringens iota toxin, C. dif f icile CDT, and C. spiroforme toxin. These toxins ADP-ribosylate G-actin in the host cells, which leads to depolymerization of the actin cytoskeleton.1 Diphtheria-, cholera-, and pertussis-toxin ADPribosylate heterotrimeric GTP-binding proteins, thereby altering the GTP-GDP-cycle. The single-chain C3-like exoenzymes are a unique class as they lack a binding and translocation domain, and their cellular uptake is not well understood so far. Several isoforms of C3 exoenzymes (also called C3 transferases) are produced by C. botulinum,2,3 Bacillus cereus,4 Staphylococcus aureus,5 and C. limosum.6 Recent studies showed that the uptake of C3 from C. botulinum depends on vimentin.7,8 C3 exoenzyme produced by Clostridium botulinum (C3bot) acts as a 24 kDa single-chain protein and consists merely of a catalytic domain that exhibits a NAD+ binding site (phosphate© 2015 American Chemical Society
nicotinamide loop) and an ADP-ribosyltransferase turn−turn loop (ARTT),9 which are both essential for ADP-ribosylation activity. C3bot transfers an ADP-ribose moiety from NAD+ to Asn-41 of the small GTPases RhoA/B/C leading to a functional inactivation of the GTPases resulting in strong morphological changes of treated cells.9 Rho acts as a master regulator of the cytoskeleton and is involved in different cellular processes such as cell motility, phagocytosis, transcription, cell division, and apoptosis.10 Furthermore, C3bot exhibits an axon and dendrite growth promoting function in primary hippocampal neurons that was independent of the catalytic activity since an inactive mutant of the C3 exoenzyme (C3E174Q) showed the same effect.11 The growth and branching promoting activity was assigned to a 29 amino acid long fragment of C3bot,12 which covers the ARTT loop. Indications for axonal regeneration and functional recovery of motor function were found in animal models.11 Nevertheless, the Received: June 27, 2015 Published: September 22, 2015 4721
DOI: 10.1021/acs.jproteome.5b00591 J. Proteome Res. 2015, 14, 4721−4733
Article
Journal of Proteome Research Protein Separation and Tryptic Digestion
mechanism of the regenerating effect in primary cells is not understood. In this study, the hippocampal cell line HT22 was used to study the impact of the enzyme-competent C3bot and a catalytically inactive mutant C3E174Q on the proteome to identify C3bot-responsive proteins. Proteins altered in their abundance after treatment of HT22 cells with C3bot should be interesting targets for further investigations on the functions of C3 exoenzymes on its growth and branching promoting effects. The proteome analysis was done using an untargeted shotgun approach with data-dependent acquisition based on the stable isotope labeling by amino acids in cell culture (SILAC) technique combined with a high-resolution liquid chromatography-mass spectrometry (LC−MS) system. A comprehensive data set was provided, which presents a detailed and kinetic overview of C3bot-responsive proteins. Multiple reaction monitoring (MRM) as a targeted approach validated the shotgun data.
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Proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) and stained with PageBlue Protein staining solution (Thermo Fisher Scientific, Germany). Twelve protein bands were manually excised and cut into 1 × 1 mm2 cubes. Gel pieces were destained with 50% acetonitrile/50 mM ammonium bicarbonate followed by dehydration with 100% acetonitrile and vacuum centrifugation. Gel pieces were rehydrated on ice for 60 min with 10% acetonitrile/20 mM ammonium bicarbonate with 5 ng/μL of trypsin and incubated at 37 °C at 300 rpm. Protein digestion was stopped for 10 min while 200 μL 50% acetonitrile/0.5% TFA was added followed by peptide extraction with 100 μL 50% acetonitrile/0.2% TFA and 100 μL 100% acetonitrile for 30 min. Supernatants were pooled, and extracted peptides were dried via vacuum centrifugation and stored at −20 °C.15,16 Liquid Chromatography
Extracted peptides were separated using a nanoflow reversed phase chromatography system (RSLC, Thermo Fisher Scientific, Germany) consisting of a trap column (2 cm length, 75 μm ID, 3 μm C18 particle) and a separating column (50 cm length, 75 μm ID, 2 μm C18 particle) (Acclaim PepMap, Thermo Fisher Scientific, Germany). For shotgun analysis, appropriate amounts of the SILAC mixture peptides were loaded, desalted, and enriched on the trap column at a flow rate of 6 μL/min of loading solvent (0.1% TFA). After 5 min, the trap column was switched in the flow of the separating column, and peptides were eluted with a multistep linear gradient of 4% mobile phase B (80% acetonitrile, 0.1% TFA) in mobile phase A (0.1% TFA) with a flow rate of 250 nL/min at 45 °C column temperature. After 10 min, the amount of mobile phase B was elevated from 4 to 25% in 95 min, 25−50% in 35 min, and 50− 90% in 5 min. This amount of mobile phase B was held constant for 10 min, decreased from 90 to 4% in 5 min, followed by equilibration of the column for 25 min. For the targeted approach, loading solvent and mobile phase A consist of 0.1% FA/0.005% TFA and mobile phase B of 80% acetonitrile/0.1% FA/0.005% TFA. The column outlet was directly connected to the nanoelectrospray source of an LTQ Orbitrap Velos (Thermo Fisher Scientific, Germany) for shotgun or a 4000 Qtrap (Sciex, Germany) mass spectrometer for targeted analyses.
EXPERIMENTAL DETAILS
Cultivation of Hippocampal Cells with SILAC Media
Murine hippocampal HT22 cells were cultivated in 75 cm2 culture flasks at 37 °C in a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) without arginine, lysine, and glutamine (PAA, Austria). The medium was supplemented with 10% dialyzed bovine fetal calf serum (Silantes, Germany), 1% (v/v) GlutaMax (Life Technologies, Germany), 1 mM sodium pyruvate, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. For comparison of three different cell states, the metabolic labeling was performed with Arg0/Lys0 (light), Arg6/Lys4 (medium), and Arg10/Lys8 (heavy) (Silantes, Germany) according to SILAC method.13 Confluent growing cells were passaged every 3−4 days in a split ratio of 1:5−1:10. Treatment of HT22 Cells and Combination of Cell Lysates
Recombinant C3bot and C3E174Q were expressed and purified from E. coli TG1 with pGEX-2T as already described.14 SILAC labeled HT22 cells were seeded in a 75 cm2 culture flask with a total amount of 3 × 106 cells. After cultivation of 24 h, cells were treated with 1 μM C3bot or C3E174Q in SILACDMEM for 24 and 48 h. SILAC-DMEM without C3 supplement was used as untreated control. Although presence of active C3 exoenzyme was proven for more than 48 h, medium supplemented with C3bot was exchanged every 48 h for the longer incubation periods of 96 and 144 h to provide fresh C3bot, nutrients, and remove metabolic products. At the same time, control cells and C3E174Q treated cells were passaged and seeded in a new culture flask. After incubation, cell morphology was documented by phase contrast microscopy (Zeiss, Germany). Subsequently, cells were washed three times with ice-cold phosphate buffered saline (PBS) and harvested in 1 mL of lysis buffer (50 mM Tris/HCl, pH 7.6, 5 mM MgCl2, 150 mM NaCl, 1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1 mM PMSF, 0.01% NP-40, ethylenediaminetetraacetic acid (EDTA) free Complete Protease Inhibitor). Experiments were performed in three biological replicates. To exclude any isotopic effect of the SILAC amino acids, a label switch was performed, and every condition was analyzed after light, medium, and heavy labeling. Differentially labeled cells were combined with same protein quantities according to different treatment.
Shotgun Proteome Analysis
For shotgun proteome analysis, peptides were analyzed with an LTQ Orbitrap Velos MS (Thermo Fisher Scientific, Germany). Eluted peptides were ionized with an emitter voltage of 1.2−1.3 kV using a metal-coated fused silica emitter. Overview scans were recorded in profile mode with a mass range from 300 to 1.600 m/z and a resolution of 60.000 at 400 m/z. On the basis of overview scans, the ten most intensive ions of charge two and three were submitted to collision-induced dissociation (CID) fragmentation using a normalized collision energy of 38%, an activation time of 10 ms, and activation Q of 0.250. Spectra were acquired with normal scan rate in centroid mode in the LTQ part of the MS. Peptides fragmented once were set on a dynamic exclusion list for 70 s to prevent further fragmentation. MS data were matched with Mascot search algorithm (Matrix Science, UK) against IPI mouse database, and peptides with a peptide ion score over 30 and a high confidence were set on a global exclusion list. This list was implemented in the instrument method, and all samples were measured twice. 4722
DOI: 10.1021/acs.jproteome.5b00591 J. Proteome Res. 2015, 14, 4721−4733
Article
Journal of Proteome Research
Figure 1. Morphological analysis of HT22 hippocampal cells after C3 treatment. HT22 cells were treated with C3bot or C3E174Q. Phase-contrast microscopy shows morphological changes after (A) 24 h of C3bot treatment. The intensity of morphological changes was enhanced after an incubation time of (B) 48 h, (C) 96 h, and (D) 144 h. Typical changes such as polynuclear and spindle-shaped cells are marked with black arrows. In contrast, no morphological changes were obvious in cells treated with C3E174Q.
Shotgun Data Processing
abundance of more than 30% (24−48 h) or 40% (96−144 h) were considered as significant regulated hits.
Shotgun raw data were processed with MaxQuant software (Version 1.2.0.18).17 Protein and peptide identification were conducted with the implemented Andromeda search engine (Version 1.2.0.12)18 and the Mus musculus IPI protein database (Version 3.79, 54 943 entries) with a false discovery rate of 0.01 at protein and peptide level. Common contaminants were excluded with the aid of an implemented database. Only proteins identified in all three biological replicates of each time point were considered. The mass tolerance for precursor ions was set to 5 ppm and to 0.7 Da for CID fragment ions. Propionamidation at cysteine residues was defined as fixed modification. Oxidation of methionine, N-terminal acetylation, and ADP-ribosylation of asparagines were set as variable modifications. Trypsin was denoted as protease with a maximum of two missed cleavages. Detection of at least two unique and razor peptide counts was required for protein identification and quantification. Protein ratios were calculated for both C3bot and C3E174Q compared to untreated control cells. Log2-transformed ratios of C3bot/control and C3E174Q/ control were subjected to a two-sided one-sample student’s t test, and proteins with a p-value
