Naunyn-Schmiedeberg's Arch Pharmacol DOI 10.1007/s00210-014-0963-7

ORIGINAL ARTICLE

Binding of Clostridium botulinum C3 exoenzyme to intact cells Astrid Rohrbeck & Leonie von Elsner & Sandra Hagemann & Ingo Just

Received: 21 January 2014 / Accepted: 12 February 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract C3 from Clostridium botulinum (C3) specifically modifies Rho GTPases RhoA, RhoB, and RhoC by monoADP-ribosylation. The confined substrate profile of C3 is the basis for its use as pharmacological tool in cell biology to study cellular functions of Rho GTPases. Although C3 exoenzyme does not possess a cell-binding/-translocation domain, C3 is taken up by intact cells via an unknown mechanism. In the present work, binding of C3 to the hippocampus-derived HT22 cells and J774A.1 macrophages was characterized. C3 bound concentrationdependent to HT22 and J774A.1 cells. Pronase treatment of intact cells significantly reduced both C3 binding and C3 cell entry. Removal of sugar residues by glycosidase F treatment resulted in an increased binding of C3, but a reduced cell entry. To explore the involvement of phosphorylation in the binding process of C3, intact HT22 and J774A.1 cells were pre-treated with vanadate prior to incubation with C3. Inhibition of de-phosphorylation by vanadate resulted in an increased binding of C3. To differentiate between intracellular and extracellular phosphorylation, intact cells were treated with CIP (calf intestine phosphatase) to remove extracellular phosphate residues. The removal of phosphate residues resulted in a strong reduction in binding of C3 to cells. In sum, the C3 membranous binding partner is proteinaceous, and the glycosylation as well as the phosphorylation state is critical for efficient binding of C3.

Electronic supplementary material The online version of this article (doi:10.1007/s00210-014-0963-7) contains supplementary material, which is available to authorized users. A. Rohrbeck (*) : L. von Elsner : S. Hagemann : I. Just Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany e-mail: [email protected]

Keywords C3 Exoenzyme . ADP-Ribosyltransferase . Overlay assay . Posttranslational modification . Glycosylation . Phosphorylation Abbreviations C3 C3 exoenzyme derived from Clostridium botulinum SDS Sodium dodecyl sulfate PAGE Polyacrylamide gel electrophoresis GlcNAc N-acetyl-D-glucosamine CIP Calf intestinal phosphatase

Introduction Clostridium botulinum C3 exoenzyme (MW 23 kDa) belongs to the family of the ADP-ribosyltransferases and modifies RhoA, RhoB, and RhoC at asparagine-41 (Vogelsgesang et al. 2007; Genth et al. 2003). This modification results in the trapping of Rho in the GDI-complex and in inhibition of Rho-GEF interaction, leading to biologically inactive (GDPbound) Rho (Sehr et al. 1998; Wilde et al. 2001; Genth et al. 2003). Inactivation of Rho causes dramatic changes in the actin cytoskeleton accompanied by rounding up of most mammalian cultured cells (Wiegers et al. 1991; Paterson et al. 1990; Rohrbeck et al. 2012). It is currently not known how C3 gets access to the target Rho proteins. C3 exoenzyme structurally lacks a translocation and binding domain. Also, the crystal structure of C3 does not give any information as to how binding to cells and uptake are mediated (Han et al. 2001). Nonspecific pinocytosis has been proposed as a mechanism of cell entry, though this required relatively high concentrations of the enzyme or extended incubation times (Aktories et al. 2000). To overcome this problem, the toxins often were introduced into target cells

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by microinjection (Paterson et al. 1990; Watanabe et al. 1997) or by the use of cell-permeable C3bot chimeras such as C3– DT, a fusion between C3 and binding domain of diphtheria toxin (Aullo et al. 1993) or C3-C2, a fusion between C3 and C2I (Barth et al. 1998). Recently, Fahrer et al. (2010) reported that C3bot and C3lim were selectively taken up by macrophages and monocytes through acidified early endosomes (Fahrer et al. 2010). This study found that an acidic pulse was sufficient to mediate direct translocation of C3 across the cell membrane, and it is argued that C3 uptake into macrophages occurs via receptor-mediated endocytosis. However, until now, this is a single report that exclusively described this finding. Many toxins such as cholera toxin (Torgersen et al. 2001) or pertussis toxin (Stein et al. 1994) interact with proteins and/ or other membrane components such as sugar moieties at the surface of target cells. As C3 modifies intracellular targets, binding to a receptor or membrane structure is an essential step for C3 in the uptake into cells. So far, neither the receptor for C3 nor the posttranslational modifications of the putative C3 receptor is known. Membrane proteins are attached to the cell membrane by crossing the membrane once or multiple times, with a sequence of hydrophobic amino acids interacting with the hydrophobic parts of the membrane. These proteins are often glycosylated at certain amino acid side chains. Protein glycosylation plays an essential role in regulating the dynamics and functions of cell surface receptors. For example, sialylation and fucosylation of epidermal growth factor receptor (EGFR) has been shown to suppress its dimerization and activation (Liu et al. 2011). The sensitivity of TGF-β receptor is regulated by N-linked glycosylation (Kim et al. 2012). Furthermore, sialylation of the β1-integrin inhibits cell adhesion to galectin-3 (Zhuo et al. 2008). C-type lectin dendritic cell immunoreceptor (DCIR) is a glycan-binding receptor present at the surface of immune cells and involved in the recognition of glycosylated pathogens and self glycoproteins (García-Vallejo and van Kooyk 2009). Recently, it was shown that glycan binding to DCIR is influenced by glycosylation of the carbohydrate recognition domain region in DCIR, and changes in the glycosylation of DCIR result in enhanced ligand binding (Bloem et al. 2013). There are lot of reports on the influence of glycosylation on protein-protein interaction at the cell surface. Phosphorylation is another important posttranslational modification of proteins, which modulates a wide range of biological functions and activities. Recently, several groups reported the identification of free phospho-proteins in different biological fluids by the use of new mass spectrometry tools (Cirulli et al. 2008; Hu et al. 2009; Zhou et al. 2009; Yalak and Vogel 2012). That extracellular phosphorylation of membrane proteins affecting the binding of ligands has not yet been reported. However, quite recently, it was demonstrated in wild-type CD11c/CD18 (monocyte/macrophage-enriched

integrin)- and S1158A CD11c/CD18-transfected K562 cells that phosphorylation at Ser-1158 regulates CD11c/CD18integrin adhesion to ligands at the cell surface (Uotila et al. 2013). The relevance of these posttranslational modifications has so far not been elucidated in C3 membrane interaction. In this study, we show for the first time that C3 binding to intact cells involves a proteinaceous structure and that this interaction is dependent on glycosylation and phosphorylation.

Materials and methods Commercially obtained reagents are as follows: glycosidase F (Roche Applied Science, Mannheim, Germany); CIP (Fermentas, Thermo Fisher Scientific Inc., Rockford, IL, USA); sodium-ortho-vanadate (Sigma-Aldrich Chemie GmbH, Munich, Germany); pronase (Roche Applied Science, Mannheim, Germany); antibodies, namely RhoA (26C4; Santa Cruz, USA), beta-actin (AC-40, SigmaAldrich Chemie GmbH, Munich, Germany), horseradish peroxidase-conjugated secondary antibody, mouse, or rabbit (Rockland Immunochemicals, Inc, Gilbertsville, PA) Cell culture Murine hippocampal HT22 cells were cultivated in Dulbecco’s modified essential medium (Biochrom; +10 % FCS, 1 % penicillin, 1 % streptomycin, and 1 mM sodium pyruvate). J774A.1 mouse macrophages were cultivated in RPMI 1640 medium (Biochrom; with 10 % FCS, 1 % penicillin, 1 % streptomycin, and 1 mM sodium pyruvate). Cells were maintained at 37 °C and 5 % CO2. Upon subconfluence, cells were passaged. Expression and purification of recombinant C3 protein C3bot was expressed as recombinant GST-fusion protein in E. coli TG1 harboring the respective DNA fragment in the plasmid pGEX-2T and purified by affinity chromatography using glutathione sepharose. The fusion protein was eluted from the beads using glutathione (Höltje et al. 2005). For cleavage of the fusion proteins from glutathione Stransferase, the beads were incubated with five “NIH” units of thrombin, and thrombin was removed by precipitation with benzamidine-sepharose beads (AP Biosciences, New York, USA). C3 binding assay For the binding assays, HT22 cells or J774A.1 cells were seeded onto 3.5 cm-plates at a concentration of 300,000 cells/ml and grown for 24 h at 37 °C and 5 % CO2.

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The medium was removed, and cells were washed with PBS. Cells were exposed 100 or 500 nM of C3 for 1 h at 4 °C. Subsequently, cells were stringent-washed three times with PBS. Cells were scraped into the Laemmli sample buffer. The obtained suspension was shaken at 37 °C for 10 min. Ultrasonic disruption was performed in a cycle of 10×5 s, 5×10 % sonic energy using a sonotrode (Bandelin Electronic, Berlin, Germany). The lysate was then incubated at 95 °C for 10 min and submitted to SDS-PAGE and Western blot analysis against C3 and β-actin. For pronase assay, we incubated intact cells with 500 μg/ml pronase for 30 min at 37 °C. Subsequently, serum was added to cells to block pronase activity, and cells were washed with PBS. C3 was then added or not, and incubation continued for further 1 h at 4 °C. To analyze sugar-dependent protein-protein interaction of C3, the intact cells were incubated with 5U/2 ml glycosidase F. After 3 h of incubation at room temperature, the cells were washed, and C3 were added for 1 h at 4 °C. Phosphorylation-dependend C3-protein interaction were analyzed by incubation of the intact cells with 50U/ml calf intestinal alkaline phosphatase for 3 or 24 h at 37 °C or by addition of 2 mM sodium-ortho-vanadate to the cells for 30 min at 37 °C. After the indicated incubation time, cells were washed, and C3 were added for 1 h at 4 °C. Western blot analysis Complete lysate proteins were separated using SDS-PAGE and subsequently transferred onto nitrocellulose membranes by a tank blot system. The membranes were blocked with 5 % (w/v) nonfat dried milk for 60 min; incubation with primary antibody was conducted overnight at 4 °C and treatment with the secondary antibody at room temperature for 1 h. For Western blot analysis, the following primary antibodies were used: RhoA was identified using a mouse monoclonal IgG from Santa Cruz Biotechnologies (Santa Cruz, USA). Identification of C3 was achieved by a rabbit polyclonal antibody (affinity purified), which was raised against the full-length toxin C3bot (accession no. CAA41767). Actin (Sigma-Aldrich Chemie GmbH, Munich, Germany) was used as loading control. For the chemiluminescence reaction, ECL Femto (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA) was used. All signals were analyzed densitometrically using the KODAK 1D software (KODAK GmbH, Stuttgart, Germany) and normalized to β-actin signals. Cell surface binding of C3-A1C/E174Q/K211C-FITC using fluorescence-activated cell sorting (FACS) For testing the interaction of C3 with cells using FACS cytometry, recombinant C3-A1C/E174Q/K211C protein was labeled with 5-flourescein isothiocyanate (FITC) microscale

protein labeling kit (AnaSpec Inc, Fremont, CA, USA) according to the manufacturer’s instructions. For the flow cytometry analysis, we used a recombinant enzyme-deficient C3-A1C/E174Q/K211C with two free amines to conjugate fluorescein-5-isothiocyanate to C3bot. C3-deficient enzyme was chosen to avoid morphological changes even after prolonged incubation time. In previous experiments, it was shown that C3 and C3-E174Q-FITC bound comparable to intact cells (Figure S1). Cells (2×105 cells/ml DMEM) were incubated at 37 °C in 5 % CO2 for 24 h. Cells were harvested in cold PBS and blocked with PBS containing 5 % FCS for 15 min on ice. Cells were washed in PBS and incubated for 60 min on ice with 500 nM C3-E174Q-FITC. Cells were washed three times with PBS, and the C3 binding was analyzed using a FACScan (FACScan flow cytometer, Becton Dickinson). Ten thousand events were monitored per condition. Reproducibility of the experiments and statistics All experiments were performed independently at least three times. Results from representative experiments are shown in the figures. Values (n≥3) are means±SEM. The two-sided unpaired Student’s t test was used throughout the study to assess the statistical significance of the differences between two sets of data. Differences were considered to be statistically significant at p≤0.05 (*=p≤0.05; **=≤0.01).

Results C3 binds to intact cells First, we examined whether C3 binds to intact hippocampal HT22 cells and to J774A.1 macrophages. Therefore, intact cells were exposed to increasing concentrations of C3 (1, 10, and 60 μM) for 1 h at 4 °C followed by stringent washing, lysis, and submission to Western blot analysis against C3bot. Figure 1a, b shows that C3 bound to both intact HT22 as well as to intact J774A.1 cells. In both cases, a concentrationdependent binding of C3 to cells was observed. Densitometric evaluation of bound C3 showed that J774A.1 cells had a higher C3-binding capacity than HT22 cells (Fig. 1c). C3 binds to protein structures at intact HT22 and J774A.1 cells To study whether a cell-surface protein was involved in the binding of C3 to cell membranes, cells were pre-incubated with pronase prior to binding to C3. Serum was added to detached cells to block pronase activity, and cells were then washed with PBS and incubated with C3 (500 nM) for 1 h at

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Fig. 1 Binding of C3 to intact murine cells. HT22 cells (a) and J774A.1 macrophage (b) cells were exposed to increasing concentrations of C3 for 1 h at 4 °C. Subsequently, cells were stringently washed three times,

lysed, and submitted to Western blot analysis against C3 and β-actin. c Densitometric evaluation of bound C3 after incubation with 10 μM of C3 (from a and b) and adjustment to the corresponding actin band are shown

4 °C. Binding of C3 to intact cells pre-treated with pronase was detected by Western blot using C3-specific antibody. Pronase treatment completely reduced the binding of C3 to intact cells (Fig. 2). Consistent with this result, treatment with pronase also resulted in a strong reduction in intracellular ADP-ribosylation of RhoA as detected by delayed or missing RhoA-shift in Western blot analysis (Fig. 3). Intracellular ADP-ribosylation of RhoA resulted in a shift in the apparent mol weight in SDS-PAGE and subsequently to degradation of modified Rho. Thus, binding and uptake of C3 into intact cells are mediated by a membranous protein.

glycosidase F prior to incubation with C3 for 1 h at 4 °C. The enzyme glycosidase F removed N-linked carbohydrates from mammalian glycoproteins. It cleaves the oligosaccharide between the GlcNAc attached to the asparagine residue of the protein (Blanchard et al. 2008). Removal of sugar residues from intact cells resulted in an increased C3 binding as shown in Western blot analysis (Fig. 4a–d). To verify this finding, a flow cytometry analysis was performed (Fig. 4e, f). Both cell lines treated with glycosidase F exhibited a significantly increased binding to C3. In the next step, we analyzed whether uptake of C3 into intact cells was dependent on carbohydrate moieties. We observed that pre-treatment of HT22 and J774A.1 intact cells with glycosidase F prior to C3 incubation caused a delayed ADP-ribosylation of RhoA in Western blot analysis. C3 in control cells caused a mol weight shift of RhoA in SDS-PAGE (based on ADP-ribosylation) and a decrease in RhoA level

C3 interaction with membranous receptor protein depends on protein glycosylation To elucidate whether glycosylation is involved in the C3protein interaction, we treated intact cells with 5U/2 ml of

Fig. 2 C3 binding to cells after pronase treatment. HT22 cells (a) and J774A.1 macrophages (b) were pre-treated with 500 μg/ml of pronase for 30 min at 37 °C. Subsequently, serum was added to cells to block pronase activity, and cells were washed with PBS. Then, C3 or PBS as control was added, and incubation continued for further 1 h at 4 °C. Cells were lysed

after washing and submitted to Western blot analysis against C3 and βactin. Western blots from representative experiments are shown (n=3). c and d Densitometric evaluation of bound C3 (from a or b) and adjustment to the corresponding actin band are shown

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Fig. 3 Internalization of C3 depends on a membrane protein. HT22 cells (a) and J774A.1 macrophages (b) were treated with 500 μg/ml of pronase for 30 min at 37 °C followed by incubation with 1 μM of C3bot for 6 h at 37 °C. After washing, cells were lysed and submitted to Western blot

analysis against RhoA and β-actin. Western blot analysis from representative experiments are shown (n=3). c and d Densitometric evaluation of ADP-ribosylated RhoA (from a or b) and adjustment to the corresponding actin band are shown

(degradation of ADP-ribosylated RhoA). In sum, extracellular carbohydrates are involved in both binding of C3 to cell membranes and cellular uptake of C3 (Fig. 5).

Discussion

C3 interaction with membranous receptor protein depends on protein phosphorylation The results from the glycosidase F assay suggest that posttranslational modifications of the surface of HT22 cells and J774A.1 macrophages play an important role in the binding of C3. To explore this hypothesis further, the involvement of protein phosphorylation in the binding to C3 was analyzed. To this end, we performed C3 binding assays after cell treatment with calf intestinal phosphatase (CIP) and vanadate, respectively. Intact cells were pre-treated with CIP (50U/ml) or vanadate (1 mM) prior to incubation with C3 for 1 h at 4 °C followed by stringently washing, lysis, and subjection to Western blot analysis against C3bot. The removal of phosphate residues by CIP resulted in a strong reduction of C3 binding (Fig. 6a–d). Conversely, inhibition of dephosphorylation by vanadate showed an increased level of C3 bound to cells (Fig. 7a–d). Taken together, treatment of the intact HT22 and J774A.1 cells with CIP or vanadate modulated the ability of cell-surface proteins to bind to C3. These data indicate that a combination of glycosylation and phosphorylation is crucial for effective interaction between C3 and the cell surface binding protein of HT22 and J774A.1 cells.

In this study, we examined the binding of C3 to intact hippocampus-derived HT22 cells and J774A.1 macrophages. The binding assays reveal a concentration-dependent binding of C3 to cells and the proteinaceous nature of the unknown binding partner of C3. Interestingly, we demonstrated that J774A.1 cells had a higher C3-binding capacity than HT22 cells. Sabri and co-workers showed that phorbol myristate acetate (PMA)-induced differentiation of THP-1 monocytes to macrophages had an effect on N-linked sugar residues (Sabri et al. 2000). For lectin, a significant higher binding capacity was determined on differentiated THP-1 cells as compared to untreated ones which might be due to the upregulation of the CD14 receptor expression during differentiation (Simmons et al. 1989). Thus, increased binding of C3 to J774A.1 macrophages might occur via these partial structures. Furthermore, posttranslational modifications such as glycosylation and phosphorylation are crucial for efficient interaction of C3 with membrane protein and the cellular uptake of C3. Extracellular domains of cell-surface receptors play essential roles in cell-cell communication, cell adhesion, and signaling pathways by binding to other proteins in the extracellular environment (Ben-Shlomo et al. 2003). To this end, extracellular and transmembrane proteins require specific posttranslational modifications. Posttranslational modifications regulate protein activity, stability, and protein-protein interaction. One of the most common protein modification is the addition of carbohydrate moieties to proteins, lipids, or

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Fig. 4 Binding of C3 to HT22 and J774A.1 cells after glycosidase F treatment. a HT22 cells were incubated with 5U/2 ml of glycosidase F and then exposed to 500 nM of C3 for 1 h at 4 °C. Subsequently, β-actin and bound C3 were detected by Western blot. One representative experiment is shown (n=3). c Densitometric evaluation of bound C3 (from a) and adjustment to the corresponding actin band are shown. Results

represent the arithmetic means±SD of three independent experiments. e Glycosidase F-treated HT22 cells were exposed to 500 nM of C3-E174QFITC for 1 h at 4 °C, and bound C3- E174Q-FITC was analyzed by FACS. (b, d, and f) same experiments for the macrophage-like cell line J774A.1. Enzymatically inactive C3-E174Q-FITC binds to intact cells comparable to wild-type C3 as shown in Fig. S1

other organic molecules inside and outside the cell. Glycosylation is site- as well as substrate-specific, tightly regulated, and reversible. Glycosylation plays a central role in protein localization and protein-protein interactions. Oligosaccharides confer resistance to proteolysis by preventing recognition by proteases and enhance the stability of the secreted molecule in vivo or in vitro (Hart et al. 2011). The surface of all cells is covered by a layer of oligosaccharides, which are the first level of interaction of cells with the environment. It has been known for years that the extent of glycosylation affects the protein-protein interaction. In fact, receptor-binding studies of human granulocyte-macrophage colony stimulating factor (hGM-CSF) demonstrated a lower receptor affinity for the heavily glycosylated form compared to the less heavily glycosylated and nonglycosylated hGM-

CSF (Cebon et al. 1990). Deglycosylated luteinizing hormone exhibits a higher receptor-binding activity than the glycosylated form (Gkonos et al. 1989). Another example for glycosylation depending on receptor binding is the observation that a higher sialylation causes a decrease in receptor binding of erythropoietin (EPO) (Darling et al. 2002). Glycosylated proteins at the cell surface of target cells are important for pathogens and bacterial toxins to interact with. For example, influenza A viruses’ hemagglutinin binds to sialylated glycans on the epithelial-cell surface (Chandrasekaran et al. 2008). The B moiety of shiga toxins binds specifically to the sugar domain of the glycosphingolipid globotriaosylceramide (Gb3) located at the plasma membrane of target cells and mediates uptake as well as intracellular transport of the toxin (Sandvig et al. 2009). For cholera toxin, it was demonstrated that

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Fig. 5 Internalization of C3 depends on membranous carbohydrate structures. HT22 cells (a) and J774A.1 macrophages (b) were treated with glycosidase F for 3 h followed by incubation with 1 μM of C3bot for 6 h at 37 °C. Cells were lysed and submitted to Western blot analysis

against RhoA and β-actin. Western blot analysis from representative experiments are shown (n=3). c and d Densitometric evaluation of ADP-ribosylated RhoA (from a or b) and adjustment to the corresponding actin band are shown

minor changes in the carbohydrate structure of the monosialoganglioside GM1 have dramatic effects on cholera toxin binding (Fishman 1982; Merritt et al. 2002). Witvliet et al. 1989 demonstrated that the optimal binding of pertussis toxin (Ptx) requires a complete sialyllactosamine moiety at surface macromolecules (Witfliet et al. 1989). Furthermore, C. botulinum type-A hemagglutinin-positive progenitor toxin binds to oligosaccharides containing Galβ1-4GlcNAc (Inoue et al. 2001). It was also shown for botulinum neurotoxin C

(BoNT/C) that the biological activity depends on ganglioside interaction (Strotmeier et al. 2011). Interestingly, we observed oligosaccharide modulated binding of C3 to cell membranes. After the removal of Nlinked carbohydrates by glycosidase F, an enhanced binding of C3 to intact hippocampal HT22 and J774A.1 cells were detected in Western blot analysis as well as in flow cytometry. Glycosidase F treatment, however, causes delayed ADPribosylation of RhoA suggesting that C3 uptake into HT22

Fig. 6 Binding of C3 to cell membranes depends on protein phosphorylation. a Intact HT22 cells pre-incubated with CIP (calf intestinal phosphatase) were incubated with 500 nM of C3 for 1 h at 4 °C. Subsequently, bound C3 and β-actin were detected by Western blot. One representative experiment is shown (n=3). b Densitometric evaluation of bound C3

(from a) and adjustment to the corresponding actin band are shown. Results are given as arithmetic means±SD from three independent experiments. c and d same experiment for the macrophage-like cell line J774A.1

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Fig. 7 Interaction of C3 with membranous receptor protein depends on stabilization of phospho-residue. HT22 cells (a) or J774A.1 macrophages (b) were pre-treated with 1 mM of vanadate for 30 min followed by incubation with 500 nM of C3 for 1 h at 4 °C. Cells were lysed and

submitted to Western blot analysis against C3 and β-actin (n=3). c–d Densitometric evaluation of bound C3 (from a or b) and adjustment to the corresponding actin band are shown. Results are given as arithmetic means±SD from three independent experiments

cells and J774A.1 macrophages is dependent on glycosylation. These findings suggest that removal of sugar residues provides more binding sites for C3, but does not accelerate uptake of C3. This observation indicates that there are possibly more than one binding site for C3. Sugars can be attached to both proteins and lipids of membranes. The composition of the glycoproteins and glycolipids varies from cell type to cell type as well as within the various intracellular compartments that are defined by intracellular membranes. However, the addition of a phosphate group to an amino acid residue (protein phosphorylation) has received considerable attention through studies of signal transduction and transcriptional regulation. Reversible protein phosphorylation is controlled through the direct action of a variety of kinases and phosphatases (Manning et al. 2002). Previously, it has been shown that phospho-proteins in yeast have more protein-protein interaction than expected by random chance (Yachie et al. 2011). The majority of the literature describes intracellular phospho-proteins and phosphorylationdependent signaling cascades. However, in the last 5 years, reports of extracellular phospho-proteins increased. It has been shown that numerous phospho-proteins are present in the extracellular space (Bahl et al. 2008; Zhou et al. 2009; Yalak and Vogel 2012). For example, Golgi-enriched-fraction casein kinase (GEF-CK) does not mediate physiological phosphorylation of casein (Duncan et al. 2000), but specifically phosphorylate peptide substrates at S-X-E/pS motifs (LasaBenito et al. 1996). This substrate motif is phosphorylated in about 75 % of human plasma and cerebrospinal fluid phospho-proteins (Salvi et al. 2010; Tagliabracci et al. 2013). Calf intestinal alkaline phosphatase (CIP), which dephosphorylates phosphoserine but not phosphothreonine residues in T antigen (Shaw and Tegtmeyer 1981; Grasser et al. 1987;

Klausing et al. 1988), was used in our study to investigate the influence of phosphorylation on C3 binding to intact cells. Indeed, a decreased binding of C3 was detected after CIP treatment of intact cells. Conversely, inhibition of protein phosphatases by vanadate causes increased binding of C3 to hippocampal HT22 cells and J774A.1 macrophages. Both results suggest that phosphorylation plays a critical role in the interaction of C3 with extracellular membrane proteins. Involvement of phosphates in the binding of C3 is conceivable as the isoelectric point of C3 (pI∼10) strongly argues for binding negatively charged phosphate groups. Posttranslational modifications occur naturally within the secretory pathway. Within the secretory pathway, proteins destined for the plasma membrane are translocated into the endoplasmic reticulum, where they are correctly folded and modified (glycosylation, phosphorylation, etc.) and then sorted into transport COPII-coated vesicles (Barlowe et al. 1994). These vesicles reach the Golgi compartment, where proteins can undergo additional modifications, such as changes of the lateral chains of some amino acids, addition or removal of sugars and other types of peptide processing. After this step, proteins are packed in secretory vesicles and directed for secretion to the plasma membrane (Kaether and Gerdes 1995; Futter et al. 1995). Therefore, it is conceivable that phospho-proteins via secretory pathway reach the cell surface. Moreover, the combination of glycosylation and phosphorylation of membrane proteins is reported. For example, the stromal interacting protein 1 (STIM1) is expressed at the cell surface, and glycosylation of STIM1 is required for cell-surface expression (Williams et al. 2002). Incorporation of [32P]orthophosphate confirmed that STIM1 protein is phosphorylated in vivo (Manji et al. 2000), and it was shown that STIM1 is located at cell surface of K562 cells and is

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phosphorylated and N-linked glycosylated. Previous work had shown that Fam20B was a kinase that phosphorylated xylose within the tetrasaccharide core region of proteoglycans (Koike et al. 2009). It is very difficult to analyse extracellular protein-protein interaction because membrane-embedded proteins are difficult to solubilize in their native conformation, and these proteins contain structurally important posttranslational modifications. The use of intact cells allows to investigate posttranslational modifications at intact cells and the effect of these posttranslational modifications on C3 proteininteraction. In this study, we show that C3 binds in a concentration-dependent manner to intact cells and that posttranslational modifications are critical for efficient interaction of C3 with membrane proteins and crucially influence the cellular uptake of C3.

Fundings This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG project JU231/5). Conflict of interest The authors declare that they have no competing interests.

References Aktories K, Schmidt G, Just I (2000) Rho GTPases as targets of bacterial protein toxins. Biol Chem 381:421–426. doi:10.1515/BC.2000.054 Aullo P, Giry M, Olsnes S, Popoff MR, Kocks C, Boquet P (1993) A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly. EMBO J 12:921– 931 Bahl JM, Jensen SS, Larsen MR, Heegaard NH (2008) Characterization of the human cerebrospinal fluid phosphoproteome by titanium dioxide affinity chromatography and mass spectrometry. Anal Chem 80:6308–6316. doi:10.1021/ac800835y Barlowe C, Orci L, Yeung T, Hosobuchi M, Hamamoto S, Salama N, Rexach MF, Ravazzola M, Amherdt M, Schekman R (1994) COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77:895–907. doi:10.1016/ 0092-8674(94)90138-4 Barth H, Hofmann F, Olenik C, Just I, Aktories K (1998) The N-terminal part of the enzyme component (C2I) of the binary Clostridium botulinum C2 toxin interacts with the binding component C2II and functions as a carrier system for a Rho ADP ribosylating C3-like fusion toxin. Infect Immun 66:1364–1369 Ben-Shlomo I, Yu Hsu S, Rauch R, Kowalski HW, Hsueh AJW (2003) Signaling receptome: a genomic and evolutionary perspective of plasma membrane receptors involved in signal transduction. Sci STKE 2003:RE9. doi:10.1126/scisignal.1872003re9 Blanchard V, Frank M, Leeflang BR, Boelens R, Kamerling JP (2008) The structural basis of the difference in sensitivity for PNGase F in the de-N-glycosylation of the native bovine pancreatic ribonucleases B and BS. Biochemistry 47:3435–3446. doi:10.1021/bi7012504 Bloem K, Vuist IM, van der Plas AJ, Knippels LM, Garssen J, GarcíaVallejo JJ, van Vliet SJ, van Kooyk Y (2013) Ligand binding and signaling of dendritic cell immunoreceptor (DCIR) is modulated by the glycosylation of the carbohydrate recognition domain. PLoS One 8:e66266. doi:10.1371/journal.pone.0066266

Cebon J, Nicola N, Ward M, Gardner I, Dempsey P, Layton J, Dührsen U, Burgess AW, Nice E, Morstyn G (1990) Granulocyte-macrophage colony stimulating factor from human lymphocytes. The effect of glycosylation on receptor binding and biological activity. J Biol Chem 265:4483–4491 Chandrasekaran A, Srinivasan A, Raman R, Viswanathan K, Raguram S, Tumpey TM, Sasisekharan V, Sasisekharan R (2008) Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin. Nat Biotechnol 26:107–113. doi:10.1038/nbt1375 Cirulli C, Chiappetta G, Marino G, Mauri P, Amoresano A (2008) Identification of free phosphopeptides in different biological fluids by a mass spectrometry approach. Anal Bioanal Chem 392:147– 159. doi:10.1007/s00216-008-2266-7 Darling RJ, Kuchibhotla U, Glaesner W, Micanovic R, Witcher DR, Beals JM (2002) Glycosylation of erythropoietin affects receptor binding kinetics: Role of electrostatic interactions. Biochemistry 41: 14524–14531. doi:10.1021/bi0265022 Duncan JS, Wilkinson MC, Burgoyne RD (2000) Purification of Golgi casein kinase from bovine milk. Biochem J 350:463–468 Fahrer J, Kuban J, Heine K, Rupps G, Kaiser E, Felder E, Benz R, Barth H (2010) Selective and specific internalization of clostridial C3 ADP-ribosyltransferases into macrophages and monocytes. Cell Microbiol 12:233–247. doi:10.1111/j.1462-5822.2009. 01393.x Fishman PH (1982) Role of membrane gangliosides in the binding and action of bacterial toxins. J Membr Biol 69:85–97 Futter CE, Connnolly CN, Cutler DF, Hopkins CR (1995) Newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell surface. J Biol Chem 270:10999–11003. doi: 10.1074/jbc.270.18.10999 García-Vallejo JJ, van Kooyk Y (2009) Endogenous ligands for C-type lectin receptors: the true regulators of immune homeostasis. Immunol Rev 230:22–37. doi:10.1111/j.1600-065X.2009. 00786.x Genth H, Gerhard R, Maeda A, Amano M, Kaibuchi K, Aktories K, Just I (2003) Entrapment of rho ADP-ribosylated by Clostridium botulinum C3 exoenzyme in the Rho-guanine nucleotide dissociation inhibitor-1 complex. J Biol Chem 278:28523–28527. doi:10. 1074/jbc.M301915200 Gkonos PJ, Charbonneau H, Lawson PT, Howard GA, Birnbaum RS, Roos BA (1989) Glycosylation reduces the bioactivity of calcitonin. Peptides 10:137–143 Grasser FA, Mann K, Walter G (1987) Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding. J Virol 61:3373–3380 Hart GW, Slawson C, Ramirez-Correa G, Lagerlof O (2011) Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease. Annu Rev Biochem 80:825–858. doi:10.1146/annurev-biochem-060608-102511 Höltje M, Hoffmann A, Hofmann F, Mucke C, Grosse G, Van Rooijen N, Kettenmann H, Just I, Ahnert-Hilger G (2005) Role of Rho-GTPase in astrocyte morphology and migratory response during in vitro wound healing. J Neurochem 5:1237–1248. doi:10.1111/j.14714159.2005.03443.x Hu L, Zhou H, Li Y, Sun S, Guo L, Ye M, Tian X, Gu J, Yang S, Zou H (2009) Profiling of endogenous serum phosphorylated peptides by titanium (IV) immobilized mesoporous silica particles enrichment and MALDI-TOFMS detection. Anal Chem 81:94–104. doi:10. 1021/ac801974f Inoue K, Fujinaga Y, Honke K, Arimitsu H, Mahmut N, Sakaguchi Y, Ohyama T, Watanabe T, Inoue K, Oguma K (2001) Clostridium botulinum type A haemagglutinin-positive progenitor toxin (HA(+)PTX) binds to oligosaccharides containing Gal beta1-4GlcNAc through one subcomponent of haemagglutinin (HA1). Microbiology 147:811–819

Naunyn-Schmiedeberg's Arch Pharmacol Kaether C, Gerdes HH (1995) Visualization of protein transport along the secretory pathway using green fluorescent protein. FEBS Lett 369: 267–271, doi.org/10.1016/0014-5793(95)00765-2 Kim YW, Park J, Lee HJ, Lee SY, Kim SJ (2012) TGF-β sensitivity is determined by N-linked glycosylation of the type II TGF-β receptor. Biochem J 445:403–411. doi:10.1042/BJ20111923 Klausing K, Scheidtmann KH, Baumann EA, Knippers R (1988) Effects of in vitro dephosphorylation on DNA-binding and DNA helicase activities of simian virus 40 large tumor antigen. J Virol 62:1258– 1265 Koike T, Izumikawa T, Tamura J, Kitagawa H (2009) FAM20B is a kinase that phosphorylates xylose in the glycosaminoglycan–protein linkage region. Biochem J 421:157–162. doi:10. 1042/BJ20090474 Lasa-Benito M, Marin O, Meggio F, Pinna LA (1996) Golgi apparatus mammary gland casein kinase: monitoring by a specific peptide substrate and definition of specificity determinants. FEBS Lett 382:149–152, doi.org/10.1016/0014-5793(96)00136-6 Liu YC, Yen HY, Chen CY, Chen CH, Cheng PF, Juan YH, Chen CH, Khoo KH, Yu CJ, Yang PC, Hsu TL, Wong CH (2011) Sialylation and fucosylation of epidermal growth factor receptor suppress its dimerization and activation in lung cancer cells. Proc Natl Acad Sci U S A 108:11332–11337. doi:10.1073/pnas.1107385108 Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298: 1912–1934. doi:10.1126/science.1075762 Merritt EA, Zhang Z, Pickens JC, Ahn M, Hol WG, Fan E (2002) Characterization and crystal structure of a high-affinity pentavalent receptor-binding inhibitor for cholera toxin and E. coli heat-labile enterotoxin. J Am Chem Soc 124:8818–8824. doi:10.1021/ ja0202560 Paterson HF, Self AJ, Garrett MD, Just I, Aktories K, Hall A (1990) Microinjection of recombinant p21rho induces rapid changes in cell morphology. J Cell Biol 111:1001–1007. doi:10.1083/jcb.111.3. 1001 Rohrbeck A, Kolbe T, Hagemann S, Genth H, Just I (2012) Distinct biological activities of C3 and ADP-ribosyltransferase-deficient C3E174Q. FEBS 279:2657–2671. doi:10.1111/j.1742-4658.2012. 08645 Sabri S, Soler M, Foa C, Pierres A, Benoliel AM, Bongrand P (2000) Glycocalyx modulation is a physiological means of regulation cell adhesion. J Cell Sci 113:1589–1600 Salvi M, Cesaro L, Tibaldi E, Pinna LA (2010) Motif analysis of phosphosites discloses a potential prominent role of the Golgi casein kinase (GCK) in the generation of human plasma phosphoproteome. J Proteome Res 9:3335–3338. doi:10.1021/pr100058r Sandvig K, Bergan J, Dyve AB, Skotland T, Torgersen ML (2009) Endocytosis and retrograde transport of Shiga toxin. Toxicon 56: 1181–1185. doi:10.1016/j.toxicon.2009.11.021. Review Sehr P, Joseph G, Genth H, Just I, Pick E, Aktories K (1998) Glucosylation and ADP-ribosylation of Rho proteins: effects on nucleotide binding, GTPase activity, and effector coupling. Biochemistry 37:5296–5304. doi:10.1021/bi972592c Shaw SB, Tegtmeyer P (1981) Binding of dephosphorylated A protein to SV40 DNA. Virology 115:88–96

Simmons DL, Tan S, Tenen DG, Nicholson-Weller A, Seed B (1989) Monocyte antigen CD14 is a phospholipid anchored membrane protein. Blood 73:284–289 Stein PE, Boodhoo A, Armstrong GD, Heerze LD, Cockle SA, Klein MH, Read RJ (1994) Structure of a pertussis toxin-sugar complex as a model for receptor binding. Nat Struct Biol 1:591–596. doi:10. 1038/nsb0994-591 Strotmeier J, Gu S, Jutzi S, Mahrhold S, Zhou J, Pich A, Eichner T, Bigalke H, Rummel A, Jin R, Binz T (2011) The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites. Mol Microbiol 81:143–156. doi:10.1111/ j.1365-2958.2011.07682.x Tagliabracci VS, Pinna LA, Dixon JE (2013) Secreted protein kinases. Trends Biochem Sci 38:121–130. doi:10.1016/j.tibs.2012.11.008 Torgersen ML, Skretting G, van Deurs B, Sandvig K (2001) Internalization of cholera toxin by different endocytic mechanisms. J Cell Sci 114:3737–3747 Uotila LM, Aatonen M, Gahmberg CG (2013) Integrin CD11c/CD18 αchain phosphorylation is functionally important. J Biol Chem 288: 33494–33499. doi:10.1074/jbc.C113.497446 Vogelsgesang M, Pautsch A, Aktories K (2007) C3 exoenzymes, novel insights into structure and action of Rho-ADP-ribosylating toxins. Naunyn Schmiedebergs Arch Pharmacol 374:347–360, Review. doi: 10.1007/s00210-006-0113-y Watanabe N, Madaule P, Reid T, Ishizaki T, Watanabe G, Kakizuka A, Saito Y, Nakao K, Jockusch BM, Naumiya S (1997) p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. EMBO J 16: 3044–3056. doi:10.1093/emboj/16.11.3044 Wiegers W, Just I, Müller H, Hellwig A, Traub P, Aktories K (1991) Alteration of the cytoskeleton of mammalian cells cultured in vitro by Clostridium botulinum C2 toxin and C3 ADP-ribosyltransferase. Eur J Cell Biol 54:237–245 Wilde C, Chhatwal GS, Schmalzing G, Aktories K, Just I. (2001) A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3. J Biol Chem 276:9537–42. doi:10.1074/jbc. M011035200 Witvliet MH, Burn DL, Brennan MJ, Poolman JT, Manclark CR (1989) Binding of pertussis toxin to eucaryotic cells and glycoproteins. Infect Immun 57:3324–3330 Yachie N, Saito R, Sugiyama N, Tomita M, Ishihama Y (2011) Integrative features of the yeast phosphoproteome and proteinprotein interaction map. PLoS Comput Biol 7:e1001064. doi:10. 1371/journal.pcbi.1001064 Yalak G, Vogel V (2012) Extracellular phosphorylation and phosphorylated proteins: not just curiosities but physiologically important. Sci Signal 5:re7, doi: 10.1126/scisignal.2003273 Zhou W, Ross MM, Tessitore A, Ornstein D, Vanmeter A, Liotta LA, Petricoin EF 3rd (2009) An initial characterization of the serum phosphoproteome. J Proteome Res 8:5523–5531. doi:10.1021/ pr900603n Zhuo Y, Chammas R, Bellis SL (2008) Sialylation of beta1 integrins blocks cell adhesion to galectin-3 and protects cells against galectin3-induced apoptosis. J Biol Chem 283:22177–22185. doi:10.1074/ jbc.M8000015200