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Complexin II regulates degranulation in RBL-2H3 cells by interacting with SNARE complex containing syntaxin-3
Cellular Immunology 261 (2010) 51–56
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Complexin II regulates degranulation in RBL-2H3 cells by interacting with SNARE complex containing syntaxin-3 Satoshi Tadokoro a, Mamoru Nakanishi b, Naohide Hirashima a,* a b
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan
a r t i c l e
i n f o
Article history: Received 6 August 2009 Accepted 22 October 2009 Available online 25 October 2009 Keywords: Mast cell Basophil Exocytosis Complexin SNARE Degranulation
a b s t r a c t Recent studies have revealed that SNARE proteins are involved in the exocytotic release (degranulation) in mast cells. However, the roles of SNARE regulatory proteins are poorly understood. Complexin is one such regulatory protein and it plays a crucial role in exocytotic release. In this study, we characterized the interaction between SNARE complex and complexin II in mast cells by GST pull-down assay and in vitro binding assay. We found that the SNARE complex that interacted with complexin II consisted of syntaxin3, SNAP-23, and VAMP-2 or -8, whereas syntaxin-4 was not detected. Recombinant syntaxin-3 binds to complexin II by itself, but its affinity to complexin II was enhanced upon addition of VAMP-8 and SNAP23. Furthermore, the region of complexin II responsible for binding to the SNARE complex, was near the central a-helix region. These results suggest that complexin II regulates degranulation by interacting with the SNARE complex containing syntaxin-3. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction In mast cells, cross-linking of high-affinity receptors for IgE (FceRI) by multivalent antigens causes activation of an intracellular signaling cascade and leads to exocytotic release of granular contents (degranulation), resulting in allergic responses [1–3]. The machinery of exocytotic release has been studied intensively in neuronal cells, and key proteins, such as soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and some of their regulatory proteins that regulate their conformation and activity, have been identified [4,5]. In mast cells, several groups including us have shown the involvement of SNARE proteins in degranulation [6–13], although the isoforms of SNARE proteins working in mast cells are different from those in neuronal cells. Recent studies have suggested that vesicle associated membrane protein VAMP-2, VAMP-7, and VAMP-8 are the possible SNARE proteins on the granule membrane (v-SNARE) in mast cells [7–12]. In these cells, SNARE proteins on the plasma membrane (t-SNARE), include syntaxin-3 and -4 and synaptosomal-associated protein 23 kDa (SNAP-23) [6–8,13]. In addition to SNARE proteins, some accessory proteins such as synaptotagmin II, Munc18-2 or -3, and complexin II are reported to be regulators of SNARE-dependent exocytosis in mast cells [14–17].
* Corresponding author. Fax: +81 52 836 3414. E-mail address: [email protected] (N. Hirashima). 0008-8749/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2009.10.011
Complexin, a soluble protein of about 15 kDa, plays a crucial role in exocytotic release, and interacts with SNARE complexes in neurons, pancreatic beta-cells, sperm cells, and mast cells [16,18–21]. Knockout or knockdown experiments of complexin suggest that it is a positive regulator of exocytotic release [16,19, 22–24]. However, the results obtained from overexpression or injection of anti-complexin antibody suggest that complexin is a negative regulator of exocytotic release [25,26]. Moreover, reconstituted liposome and cell–cell fusion assays also suggest that complexin acts as a fusion clamp that is released by Ca2+-bound synaptotagmin [27–30]. In congruence with the results that distinct domains of complexin differentially regulate exocytotic release [31], the physiological role of complexin is still controversial. Previously, we reported that complexin II, and not complexin I, is expressed in mast cells, and its knockdown using the anti-sense technique inhibited degranulation [16]. These results indicated that complexin II positively regulates degranulation in mast cells. However, the molecular mechanism of how complexin II regulates SNARE-dependent exocytosis by complexin II is unclear. In neuronal cells, it is widely known that complexin binds the SNARE complex immediately before exocytotic release. Therefore, it would be of great interest to determine the SNARE proteins that are regulated by complexin II in mast cells. In this study, we investigated the SNARE-binding partner of complexin in mast cells. To address this problem, we performed a glutathione S-transferase (GST) pulldown assay and an in vitro binding assay using recombinant SNARE proteins. Furthermore, we attempted to determine the
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region of complexin II that is responsible for binding to the SNARE complex in mast cells. 2. Materials and methods
total cell lysate of RBL-2H3 and incubated at 4 °C. After washing with His wash buffer [25 mM HEPES–KOH (pH 7.4), 1% Triton X100, 400 mM KCl, 2 mM b-mercaptoethanol, 10% glycerol, 50 mM imidazole], the eluate was subjected to SDS–PAGE and Western blot analysis.
2.1. Cell culture 2.6. Western blotting RBL-2H3 cells were cultured in Eagle’s minimal essential medium (Nissui, Tokyo, Japan) with 10% fetal bovine serum (GIBCO, Grand Island, NY) at 37 °C in a humidified atmosphere with 5% CO2. 2.2. Expression and purification of recombinant proteins Full-length rat syntaxin-3, VAMP-8, and SNAP-23 were expressed in Escherichia coli, and purified as described previously [32]. Full-length rat complexin II gene and rat complexin II deletion mutants were ligated into pGEX-4T 1 vector (Amersham Pharmacia Biotech, Uppsala, Sweden). The plasmids were then introduced into BL21 Star E. coli (Invitrogen, Carlsbad, CA). Transformed cells were grown in LB medium to an OD600 of 0.8, and protein expression was induced by 1 mM isopropyl b-D-L-thiogalactopyranoside (IPTG) treatment for 3 h at 37 °C. GST-complexin II was purified by MagneGST Protein Purification System (Promega, Madison, WI). In some experiments, GST was cleaved by thrombin. 2.3. Complexin II deletion mutants The primer pairs used to amplify complexin II lacking either the C-terminus (the region from residues 1–88) or the N-terminus (the region from residues 41–134), were 50 -GGATCCATGGACTTCGTC AT-30 (sense)/50 -CTCGAGCTGTTCCAGGGCTGCCTTCT-30 (anti-sense) and 50 -GGATCCCTGAGGCAGCAGGAGGAAGA-30 (sense)/50 -GCGGCC GCTTACTTCTTGAACATGTCCTGCA-30 (anti-sense), respectively. The obtained PCR fragments encoding complexin II lacking either the C-terminus or the N-terminus were subcloned into pGEX-4T 1 vector (Amersham Pharmacia Biotech). 2.4. Site-directed mutagenesis To obtain mutant complexin II(R59H), mutated complexin II in which CGT for Arg59 is substituted with CAT for His was prepared by the overlap extension method, using two pairs of oligonucleotides (50 -CCATGGACTTCGTCATGAAGCA-30 (sense)/50 -GGACCTTCTC ATGTTCCGCTTCCA-30 (anti-sense) and 50 -TGGAAGCGGAACATGAG AAGGTCC-30 (sense)/50 -GGATCCCTTCTTGAACATGTCCTGCA-30 (antisense), mutated codons are underlined. The obtained PCR fragment encoding the mutant complexin II(R59H) was subcloned into the NcoI–BamHI site of pQE60 (Qiagen, Hilden, Germany) and verified by DNA sequencing. 2.5. Pull-down assay A GST pull-down assay was performed using a GST Protein Interaction Pull-Down Kit (Pierce, Rockford, IL). In brief, cell lysate obtained from E. coli expressing GST-complexin II was mixed with glutathione agarose beads. GST-complexin II bound to glutathione beads was mixed with total cell lysate of RBL-2H3 and incubated at 4 °C. After washing with wash solution [a 1:1 mix of Tris-buffered saline (TBS) to ProFound Lysis Buffer (Pierce)], elution buffer was added and the eluate was subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis. For His pull-down assay, cell lysate obtained from E. coli expressing complexin II-His or complexin II(R59H)-His was mixed with Ni–NTA agarose beads (Qiagen). Complexin II-His or complexin II(R59H) bound to Ni–NTA agarose beads was mixed with
SDS–PAGE and Western blot analysis were performed as described previously [16]. Primary antibodies used are as follows: rabbit anti-SNAP-23 antibody (dilution 1:1000; Synaptic Systems, Göttingen, Germany), rabbit anti-syntaxin-3 antibody (1:1000; Synaptic Systems), rabbit anti-syntaxin-4 antibody (1:1000; Synaptic Systems), rabbit anti-VAMP-8 antibody (1:1000; Synaptic Systems), mouse anti-VAMP-7 antibody (1:100; Upstate Biotechnology, Lake Placid, NY), mouse anti-VAMP-2 antibody (1:2000; Synaptic Systems), mouse anti-Munc18 antibody (1:1000; BD Bioscience Pharmingen, San Diego, CA), and goat anti-synaptotagmin II (1:200; Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were detected by enhanced chemiluminescence (SuperSignal West Pico, Pierce) using a luminescence analyzer (LAS-3000 mini; FUJI FILM, Tokyo, Japan). 2.7. In vitro binding assay All reactions were performed in 400 ll binding buffer [50 mM Tris–HCl (pH 8.0), 100 mM KCl, 10% glycerol, 10 mM b-mercaptoethanol, and 0.8% b-octyl glucoside]. A sample of 3 lg GST-complexin II was immobilized on 20 ll of glutathione beads. SNARE proteins were added and the reaction mixture was maintained at 4 °C for 1 h. After washing with the binding buffer, sample buffer was added and eluted proteins were subjected to SDS–PAGE and Western blot analysis. 3. Results 3.1. Characterization of the SNARE complex that interacts with complexin II We tried to identify isoforms of SNAREs that interact with complexin II, and clarify the relationship between complexin II and other accessory SNARE proteins in mast cells. For this purpose, we obtained GST or GST-complexin II recombinant protein from transformed E. coli. These recombinant proteins were separated by SDS–PAGE and stained by Coomassie Brilliant Blue (CBB) to confirm their expression and purity. To determine the components of the SNARE complex that interact with complexin II in mast cells, a GST pull-down assay was carried out, and coprecipitated components were subjected to Western blot analysis (Fig. 1). We found that the SNARE complex that interacts with complexin II consisted of syntaxin-3, SNAP-23, VAMP-2, or VAMP-8. On the other hand, syntaxin-4 was not detected in the pull-down complex. We also investigated whether or not SNARE accessory proteins, such as Munc18-2 and synaptotagmin II, were present in this pull-down complex, but these proteins were not detected. This might be because Munc18 favors free syntaxin, and synaptotagmin competes with complexin for binding to the SNARE complex [30,33]. 3.2. Molecular interaction between complexin II and SNARE proteins We examined the binding affinity of complexin II to SNARE proteins to understand their binding. GST-SNAP-23, syntaxin-3-His, GST-VAMP-8, and GST-complexin II were expressed in E. coli and purified with affinity chromatography. Purified proteins were
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Fig. 1. SNARE proteins interacting with complexin II in mast cells. SNARE proteins that interact with complexin II were identified by GST pull-down assay. As a negative control, GST alone bound to glutathione beads was mixed with total cell lysates of RBL-2H3. Interacting proteins were detected by Western blot analysis. As a positive control, 3 lg total cell lysates of RBL-2H3 were loaded (n = 5).
electrophoresed by SDS–PAGE and stained with CBB (Fig. 2A). The purified GST-complexin II bound to glutathione beads was mixed with recombinant SNARE proteins. After washing with wash solution, the elution buffer was added and bound materials were subjected to SDS–PAGE and Western blot analysis (Fig. 2B). VAMP-8 and syntaxin-3 was able to bind to GST-complexin II by themselves, but SNAP-23 could not. Syntaxin-3 was able to bind to complexin II as well by itself. The binding affinity of complexin II with syntaxin-3 was enhanced in the presence of VAMP-8 (0.13 lg/ 400 ll) and SNAP-23 (0.13 lg/400 ll) to syntaxin-3. These results suggest that complexin II binds to the SNARE complex with a higher affinity than VAMP-8 or syntaxin-3 alone.
3.3. Binding region of complexin II to the SNARE complex Next, we tried to determine the region of complexin II that is necessary for binding to the SNARE complex in mast cells. To address this problem, we obtained the 1–88 aa mutant that lacks the C-terminal amino acids 89–134, and the 41–134 aa mutant that lacks the N-terminal amino acids 1–40. We also prepared a point-mutated complexin II (R59H). Full-length and two deletion mutants were tagged with GST. Full-length and mutated complexin IIR59H were tagged with hexahistidine. Pull-down assay was carried out using these proteins. The pull-down complex was subjected to either SDS–PAGE followed by Western blot analysis (Fig. 3A, upper panel) or stained by CBB (Fig. 3A, lower panel). CBB staining revealed that approximately equal amounts of full-length and both deletion mutants were immobilized on glutathione beads. Both deletion mutants of complexin II bound to approximately equal amounts of SNARE proteins compared with full-length complexin II. On the other hand, complexin IIR59H reduced its binding to the SNARE complex (Fig. 3B). Probably the site chain of histidine is exhibited shorter than that of arginine that serves for a salt bridge with aspartic acid residue of VAMP-2 [34]. The aspartic acid of VAMP-2 corresponds to asparagine in VAMP-8, and this might explain the binding to complexin II with a lower affinity. These results demonstrate that the central a-helix region, especially Arg59, is responsible for binding to SNARE complex in mast cells.
Fig. 2. In vitro assay of interaction between SNARE proteins and complexin II. (A) GST-SNAP-23, syntaxin-3-His, GST-VAMP-8, and GST-complexin II were expressed in E. coli and purified. Samples were separated by SDS–PAGE and stained by CBB. (B) GST-complexin II (3 lg) bound to glutathione beads was mixed with one of SNAREs (SNAP-23, VAMP-8 and syntaxin-3) at indicated concentrations except for the lowest frame (SNAP-23 + Syntaxin-3 + VAMP-8) in which GST-complexin II was mixed with all of SNAREs (SNAP-23 (0.13 lg/400 ll) + Syntaxin-3 (indicated concentration) + VAMP-8 (0.13 lg/400 ll)). Pull-down samples were subjected to SDS–PAGE and Western blot analysis. When GST-complexin II were mixed with one of SNAREs, immunoreactivity was detected with a specific antibody for each SNAREs (upper three frames). When GST-complexin II were mixed with all of SNAREs, immunoreactivity was detected with a specific antibody for syntaxin-3 (the lowest frame). The binding affinity of complexin II with syntaxin-3 was enhanced in the presence of VAMP-8 (0.13 lg) and SNAP-23 (0.13 lg) to syntaxin-3 (n = 4).
4. Discussion We previously reported that complexin II is expressed in mast cells and the knockdown of complexin II inhibited degranulation. Moreover, we found that the knockdown of complexin II decreased Ca2+ sensitivity of the degranulation machinery [16]. However, it was not clear which SNARE proteins were regulated by complexin II. In this study, we investigated the SNARE complex that is regulated by complexin II in mast cells. The results of the pull-down assay and the in vitro binding assay suggest that the SNARE complex containing syntaxin-3 is regulated by complexin II in mast cells. As shown in Fig. 2B, complexin II binds to syntaxin-3, and this binding was enhanced by addition of SNAP-23 and VAMP-8 (bottom panel). Although we have not direct evidence, the binding enhancement seems to be due to the formation of a stable SNARE complex comprised of SNAP-23, syntaxin-3, VAMP-8 and complexin II. If syntaxin-3 and VAMP-8 bind to complexin II at the same biding site of complexin II, binding of syntaxin-3 to complexin II would be decreased in the presence of VAMP-8. However, bound syntaxin-3 was increased in the presence of VAMP-8, sug-
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Fig. 3. Binding analysis of complexin II with SNAREs. Deletion mutants of complexin II (1–88 amino acids and 41–134 amino acids) were tagged with GST and immobilized on glutathione beads. As positive and negative controls, GSTcomplexin II and GST were used, respectively. These immobilized proteins were mixed with total cell lysates of RBL-2H3, and pull-down samples were subjected to SDS–PAGE and Western blot analysis (A, upper panel) or stained by CBB (A, lower panel) (n = 3). Complexin IIR59H was tagged with hexahistidine and immobilized Ni– NTA agarose beads. As a positive control, hexahistidine-tagged complexin II was used. These immobilized proteins were mixed with total cell lysates of RBL-2H3. As a negative control, Ni–NTA agarose beads alone was mixed with them. Pull-down samples were subjected to SDS–PAGE and Western blot analysis (B). Amounts of SNAP-23, syntaxin-3, VAMP-2 and VAMP-8 bound to complexin IIR59H decreased to 10.1 ± 2.6%, 35.3 ± 4.3%, 35.5 ± 5.2% and 58.9 ± 8.5% of control, respectively (n = 4).
gesting that the binding site is different each other and both syntaxin-3 and VAMP-8 are included in the complex. SNAP-23 did not bind to complexin II directly, but similar GST pull-down experiments using neural SNAREs have shown that SNAP-25 enhances binding of syntaxin-1 to complexin II by forming SNARE complex, while VAMP-2 has little effects [18,21]. Therefore, it is probable that the complex formed in the experiment shown in the bottom panel in Fig. 2B includes SNAP-23 as well as VAMP-8. Moreover, complexin II might interact with the SNARE complex across syntaxin and VAMP, since both syntaxin-3 and VAMP-8 can bind to complexin II by themselves. This configuration also agrees with the previous identification of crystal structure [34]. Pabst et al. investigated the association of complexin with the SNARE complex using different isoforms of syntaxin [35,36]. They showed that complexin binds to a SNARE complex comprising SNAP-25, VAMP-2, and syntaxin-3 as efficiently as it binds to complex com-
prised of SNAP-25, VAMP-2 and syntaxin-1. However, complexin did not bind to the SNARE complex containing syntaxin-4. Moreover, Reim et al. also reported that complexin II interacts with a SNARE complex comprising SNAP-25, VAMP-2, syntaxin-3 at retinal ribbon synapses [37]. These results suggest that complexin II binds to the SNARE complex through syntaxin-3 but not through syntaxin-4. As for VAMP, we found that complexin II interacted with SNARE complex containing VAMP-2 or -8 in mast cells. Recently, Malsam et al. reported that complexin I interacts with SNAP-25/syntaxin-1/VAMP-2 or -8 and affects the fusion of syntaxin-1/SNAP-25 liposome with VAMP-8 and VAMP-2 liposome [38]. Taking these results into account, complexin II seems to regulate the SNARE complex containing syntaxin-3 regardless of the subtype of VAMP. We also determined the region of complexin II that is necessary for binding to the SNARE complex in mast cells. Chen et al. reported that complexin I binds to the SNARE complex, which consists of syntaxin-1, SNAP-25, VAMP-2, through a central a-helix (the region between residue 48 and 70 of complexin I) and Arg59 in complexin contributes to crucial contacts with SNARE complex [34]. Moreover, point mutation of complexin (R59H) decreased SNARE complex binding and abolished its overexpression effects in neuron [39]. In mast cell, the central a-helix of complexin II is also important for binding to SNARE complex and complexin (R59H) may be able to use as a dominant negative form. In mast cells, Paumet et al. showed that syntaxin-4, but not syntaxin-3, immunoprecipitated with SNAP-23 [7]. They also found that overexpression of syntaxin-4 inhibited degranulation, whereas over expression of syntaxin-3 had not such effect [7]. Moreover, injection of anti-syntaxin-4 antibody caused inhibition of degranulation [12]. These results suggest that syntaxin-4 rather than syntaxin-3 is responsible for degranulation in mast cells. In contrast, we found that complexin II regulates degranulation in mast cells and binds to the SNARE complex through interaction with syntaxin-3 and not with syntaxin-4, suggesting the involvement of syntaxin-3 in degranulation. Several lines of evidence support this possibility. First, the cis-SNARE complex is formed from SNAP-23, syntaxin-3, and VAMP-7 [9]. Second, Munc18-2, one of the SNARE accessory proteins, regulates degranulation by interaction with syntaxin-3 in mast cells [14,15]. Third, in the SNARE reconstituted liposome fusion assay, syntaxin-3 as well as syntaxin-4 together with SNAP-23 and VAMP-8, is enough to induce membrane fusion [32]. As for isoforms of syntaxin involved in exocytosis in mast cells, both syntaxin-3 and -4 might be involved. One possible difference between them in the regulation of exocytosis is that syntaxin-3 dependent process is regulated by SNARE accessory proteins such as munc18-2 and complexin II, while syntaxin-4 dependent process is not [14–16]. Another point that might explain the involvement of two isoforms of syntaxin is that there are different types of secretory granules that utilize different syntaxin to fuse with the plasma membrane. Although mast cell is not a polar cell, there might exist distinct regions in the plasma membrane where either syntaxin-3 or -4 are enriched. In the case of MDCK (Madin-Darby canine kidney) cells, syntaxin-3 is localized in the apical plasma membrane and syntaxin-4 is localized in the basolateral plasma membrane. In these cells, syntaxin-3 and -4 are differently involved in membrane traffic to apical and basolateral membrane [40,41]. For isoforms of VAMP, VAMP-8 plays a major role in the degranulation since VAMP-2-deficient BMMC (bone marrow-derived mast cells) did not exhibit defects in degranulation [11]. Fig. 4 depicts the role of complexin II and SNAREs in the exocytotic membrane fusion. Complexin II interacts with SNARE complex through binding to syntaxin-3 and VAMP-8 with its central a-helical region. Syntaxin-4 also forms SNARE complex with SNAP-23 and VAMP-8, but complexin II does not seem to regulate this type of SNARE complex. As we reported previously, bind-
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Fig. 4. Schematic representation of regulation mechanism by complexin II and SNAREs in degranulation. SNAP-23 and syntaxin-3 are expressed on the plasma membrane. VAMP-8 is expressed on granular membranes. After stimulation, SNAP-23, syntaxin-3 and VAMP-8 assemble and form SNARE complex. Complexin II binds to this SNARE complex through syntaxin-3 and VAMP-8, stabilizing the complex. Sustained increase in intracellular Ca2+ concentration causes exocytotic membrane fusion. Complexin II may be released from the complex after membrane fusion as in neuronal cells. Although syntaxin-4 is also expressed and forms SNARE complex, complexin II does not bind to this type of SNARE complex.
ing of complexin II to SNARE complex alone does not trigger exocytotic membrane fusion [16]. It requires sustained increase in intracellular Ca2+ by Ca2+ influx from extracellular medium. In some types of secretory cells including mast cells, a membrane fusion between secretory granules is also observed [42,43]. It is believed that granule–granule fusion allows granules far from the plasma membrane to secrete intragranular contents. We previously reported that syntaxin-3 is found not only on the plasma membrane but also on the secretory granules in mast cells [8,15]. Therefore, syntaxin-3 on the secretory granule might be involved in both the fusion with the plasma membrane and granule–granule fusion to facilitate exocytotic release. Acknowledgments This work was supported in part by the Ministry of Education, Culture, Sports, and Technology of Japan (Grants 17049024 and 18370064 to N.H.), and Grant-in-Aid for Research in Nagoya City University to S.T. References [1] S.T. Holgate, The epidemic of allergy and asthma, Nature 402 (1999) B2–B4. [2] H. Turner, J.P. Kinet, Signaling through the high-affinity IgE receptor EceRI, Nature 402 (1999) B24–B30. [3] U. Blank, J. Rivera, The ins and outs of IgE-dependent mast-cell exocytosis, Trends Immunol. 25 (2004) 266–273. [4] T.H. Sollner, Regulated exocytosis and SNARE function, Mol. Membr. Biol. 20 (2003) 209–220. [5] Y.A. Chen, R.H. Scheller, SNARE-mediated membrane fusion, Nat. Rev. Mol. Cell Biol. 2 (2000) 98–106. [6] Z. Guo, C. Turner, D. Castle, Relocation of the t-SNARE SNAP-23 from lamellipodia-like cell surface projections regulates compound exocytosis in mast cells, Cell 94 (1998) 537–548. [7] F. Paumet, J. Le Mao, S. Martin, T. Galli, B. David, U. Blank, M. Roa, Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8 containing secretory compartment, J. Immunol. 164 (2000) 5850– 5857. [8] T. Hibi, N. Hirashima, M. Nakanishi, Rat basophilic leukemia cells express syntaxin-3 and VAMP-7 in granule membranes, Biochem. Biophys. Res. Commun. 271 (2000) 36–41. [9] N. Puri, M.J. Kruhlak, S.W. Whiteheart, P.A. Roche, Mast cell degranulation requires N-ethylmaleimide-sensitive factor-mediated SNARE disassembly, J. Immunol. 171 (2003) 5345–5352. [10] N. Tiwari, C.C. Wang, C. Brochetta, G. Ke, F. Vita, Z. Qi, J. Rivera, M.R. Soranzo, G. Zabucchi, W. Hong, U. Blank, VAMP-8 segregates mast cell-preformed mediator exocytosis from cytokine trafficking pathways, Blood 111 (2008) 3665–3674.
[11] N. Puri, P.A. Roche, Mast cells possess distinct secretory granule subsets whose exocytosis is regulated by different SNARE isoforms, PNAS 105 (2008) 2580– 2585. [12] L.E. Sander, S.P. Frank, S. Bolat, U. Blank, T. Galli, H. Bigalke, S.C. Bischoff, A. Lorentz, Vesicle associated membrane protein (VAMP)-7 and VAMP-8, but not VAMP-2 or VAMP-3, are required for activation-induced degranulation of mature human mast cells, Eur. J. Immunol. 38 (2008) 855–863. [13] N. Puri, P.A. Roche, Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis, Traffic 11 (2006) 1482–1494. [14] S. Martin-Verdeaux, I. Pombo, B. Iannascoli, M. Roa, N. Varin-Blank, J. Rivera, U. Blank, Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis, J. Cell Sci. 116 (2003) 325–334. [15] S. Tadokoro, T. Kurimoto, M. Nakanishi, N. Hirashima, Munc18-2 regulates exocytotic membrane fusion positively interacting with syntaxin-3 in RBL2H3 cells, Mol. Immunol. 44 (2007) 3427–3433. [16] S. Tadokoro, M. Nakanishi, N. Hirashima, Complexin II facilitates exocytotic release in mast cells by enhancing Ca2+ sensitivity of the fusion process, J. Cell Sci. 118 (2005) 2239–2246. [17] D. Baram, R. Adachi, O. Medalia, M. Tuvim, B.F. Dickey, Y.A. Mekori, R. SagiEisenberg, Synaptotagmin II negatively regulates Ca2+-triggered exocytosis of lysosomes in mast cells, J. Exp. Med. 189 (1999) 1649–1658. [18] H.T. McMahon, M. Missler, C. Li, T.C. Südhof, Complexins: cytosolic proteins that regulate SNAP receptor function, Cell 83 (1995) 111–119. [19] A. Abderrahmani, G. Niederhauser, V. Plaisance, M.E. Roehrich, V. Lenain, T. Coppola, R. Regazzi, G. Waeber, Complexin I regulates glucose-induced secretion in pancreatic beta-cells, J. Cell Sci. 117 (2004) 2239–2247. [20] C.M. Roggero, G.A. De Blas, H. Dai, C.N. Tomes, J. Rizo, L.S. Mayorga, Complexin/ synaptotagmin interplay controls acrosomal exocytosis, J. Biol. Chem. 282 (2007) 26335–26343. [21] H. Tokumaru, K. Umayahara, L.L. Pellegrini, T. Ishizuka, H. Saisu, H. Betz, G.J. Augustine, T. Abe, SNARE complex oligomerization by synaphin/complexin is essential for synaptic vesicle exocytosis, Cell 104 (2001) 421–432. [22] K. Reim, M. Mansour, F. Varoqueaux, H.T. McMahon, T.C. Südhof, N. Brose, C. Rosenmund, Complexins regulate a late step in Ca2+-dependent neurotransmitter release, Cell 104 (2001) 71–81. [23] H. Cai, K. Reim, F. Varoqueaux, S. Tapechum, K. Hill, J.B. Sorensen, N. Brose, R.H. Chow, Complexin II plays a positive role in Ca2+-triggered exocytosis by facilitating vesicle priming, PNAS 105 (2008) 19538–19543. [24] M. Xue, A. Stradomska, H. Chen, N. Brose, W. Zhang, C. Rosenmund, K. Reim, Complexins facilitate neurotransmitter release at excitatory and inhibitory synapses in mammalian central nervous system, PNAS 105 (2008) 7875–7880. [25] S. Ono, G. Baux, M. Sekiguchi, P. Fossier, N.F. Morel, I. Nihonmatsu, K. Hirata, T. Awaji, S. Takahashi, M. Takahashi, Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals, Eur. J. Neurosci. 10 (1998) 2143–2152. [26] M. Itakura, H. Misawa, M. Sekiguchi, S. Takahashi, M. Takahashi, Transfection analysis of functional roles of complexin I and II in the exocytosis of two different types of secretory vesicles, Biochem. Biophys. Res. Commun. 265 (1999) 691–696. [27] C.G. Giraudo, W.S. Eng, T.J. Melia, J.E. Rothman, A clamping mechanism involved in SNARE-dependent exocytosis, Science 313 (2006) 676–680. [28] J.R. Schaub, X. Lu, B. Doneske, Y.K. Shin, J.A. McNew, Hemifusion arrest by complexin is relieved by Ca2+-synaptotagmin I, Nat. Struct. Mol. Biol. 13 (2006) 748–750. [29] T. Melia Jr, Putting the clamps on membrane fusion: how complexin sets the stage for calcium-mediated exocytosis, FEBS Lett. 581 (2007) 2131–2139.
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[30] J. Tang, A. Maximov, O.H. Shin, H. Dai, J. Rizo, T.C. Südhof, A complexin/ synaptotagmin 1 switch controls fast synaptic vesicle exocytosis, Cell 126 (2006) 1175–1187. [31] M. Xue, K. Reim, X. Chen, H.T. Chao, H. Deng, N. Brose, C. Rosenmund, Distinct domains of complexin I differentially regulate neurotransmitter release, Nat. Struct. Mol. Biol. 14 (2007) 949–958. [32] H. Sakiyama, S. Tadokoro, M. Nakanishi, N. Hirashima, Membrane fusion between liposomes containing SNARE proteins involved in mast cell exocytosis, Inflamm. Res. 58 (2009) 139–142. [33] Y. Hata, C.A. Slaughter, T.C. Südhof, Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin, Nature 366 (1993) 347–351. [34] X. Chen, D.R. Tomchick, E. Kovrigin, D. Araç, M. Machius, T.C. Südhof, J. Rizo, Three-dimensional structure of the complexin/SNARE complex, Neuron 33 (2002) 397–409. [35] S. Pabst, J.W. Hazzard, W. Antonin, T.C. Südhof, R. Jahn, J. Rizo, D. Fasshauer, Selective interaction of complexin with the neuronal SNARE complex, determination of the binding regions, J. Biol. Chem. 275 (2000) 19808–19818. [36] M. Pabst, D. Vainius, R. Langen, R. Jahn, D. Fasshauer, Rapid and selective binding to the synaptic SNARE complex suggests a modulatory role of complexions in neuroexocytosis, J. Biol. Chem. 277 (2000) 7838–7848.
[37] K. Reim, H. Wegmeyer, J.H. Brandstätter, M. Xue, C. Rosenmund, T. Dresbach, K. Hofmann, N. Brose, Structurally and functionally unique complexins at retinal ribbon synapses, J. Cell Biol. 169 (2005) 669–680. [38] J. Malsam, F. Seiler, Y. Schollmeier, P. Rusu, J.M. Krause, T.H. Sollner, The carboxy-terminal domain of complexin I stimulates liposome fusion, PNAS 106 (2009) 2001–2006. [39] D.A. Archer, M.E. Grraham, R.D. Burgoyne, Complexin regulates the closure of the fusion pore during regulated vesicle exocytosis, J. Biol. Chem. 24 (2002) 18249–18252. [40] F. Lafont, P. Verkade, T. Galli, C. Wimmer, D. Louvard, K. Simons, Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells, PNAS 96 (1999) 3734–3738. [41] S.H. Low, S.J. Chapin, C. Wimmer, S.W. Whiteheart, L.G. Kömüves, K.E. Mostov, T. Weimbs, The SNARE machinery is involved in apical plasma membrane trafficking in MDCK cells, J. Cell Biol. 141 (1998) 1503–1513. [42] Y. Kawasaki, T. Saitoh, T. Okabe, K. Kumakura, M. Ohara-Imaizumi, Visualization of exocytotic secretory processes of mast cell by fluorescence techniques, Biochem. Biophys. Acta 1067 (1991) 71–80. [43] T. Kishimoto, R. Kimura, T.T. Liu, T. Nemoto, Vacuolar sequential exocytosis of large dense-core vesicles in adrenal medulla, EMBO J. 25 (2006) 673–682.
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Complexin II regulates degranulation in RBL-2H3 cells by interacting with SNARE complex containing syntaxin-3 Satoshi Tadokoro a, Mamoru Nakanishi b, Naohide Hirashima a,* a b
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan
a r t i c l e
i n f o
Article history: Received 6 August 2009 Accepted 22 October 2009 Available online 25 October 2009 Keywords: Mast cell Basophil Exocytosis Complexin SNARE Degranulation
a b s t r a c t Recent studies have revealed that SNARE proteins are involved in the exocytotic release (degranulation) in mast cells. However, the roles of SNARE regulatory proteins are poorly understood. Complexin is one such regulatory protein and it plays a crucial role in exocytotic release. In this study, we characterized the interaction between SNARE complex and complexin II in mast cells by GST pull-down assay and in vitro binding assay. We found that the SNARE complex that interacted with complexin II consisted of syntaxin3, SNAP-23, and VAMP-2 or -8, whereas syntaxin-4 was not detected. Recombinant syntaxin-3 binds to complexin II by itself, but its affinity to complexin II was enhanced upon addition of VAMP-8 and SNAP23. Furthermore, the region of complexin II responsible for binding to the SNARE complex, was near the central a-helix region. These results suggest that complexin II regulates degranulation by interacting with the SNARE complex containing syntaxin-3. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction In mast cells, cross-linking of high-affinity receptors for IgE (FceRI) by multivalent antigens causes activation of an intracellular signaling cascade and leads to exocytotic release of granular contents (degranulation), resulting in allergic responses [1–3]. The machinery of exocytotic release has been studied intensively in neuronal cells, and key proteins, such as soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and some of their regulatory proteins that regulate their conformation and activity, have been identified [4,5]. In mast cells, several groups including us have shown the involvement of SNARE proteins in degranulation [6–13], although the isoforms of SNARE proteins working in mast cells are different from those in neuronal cells. Recent studies have suggested that vesicle associated membrane protein VAMP-2, VAMP-7, and VAMP-8 are the possible SNARE proteins on the granule membrane (v-SNARE) in mast cells [7–12]. In these cells, SNARE proteins on the plasma membrane (t-SNARE), include syntaxin-3 and -4 and synaptosomal-associated protein 23 kDa (SNAP-23) [6–8,13]. In addition to SNARE proteins, some accessory proteins such as synaptotagmin II, Munc18-2 or -3, and complexin II are reported to be regulators of SNARE-dependent exocytosis in mast cells [14–17].
* Corresponding author. Fax: +81 52 836 3414. E-mail address: [email protected] (N. Hirashima). 0008-8749/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2009.10.011
Complexin, a soluble protein of about 15 kDa, plays a crucial role in exocytotic release, and interacts with SNARE complexes in neurons, pancreatic beta-cells, sperm cells, and mast cells [16,18–21]. Knockout or knockdown experiments of complexin suggest that it is a positive regulator of exocytotic release [16,19, 22–24]. However, the results obtained from overexpression or injection of anti-complexin antibody suggest that complexin is a negative regulator of exocytotic release [25,26]. Moreover, reconstituted liposome and cell–cell fusion assays also suggest that complexin acts as a fusion clamp that is released by Ca2+-bound synaptotagmin [27–30]. In congruence with the results that distinct domains of complexin differentially regulate exocytotic release [31], the physiological role of complexin is still controversial. Previously, we reported that complexin II, and not complexin I, is expressed in mast cells, and its knockdown using the anti-sense technique inhibited degranulation [16]. These results indicated that complexin II positively regulates degranulation in mast cells. However, the molecular mechanism of how complexin II regulates SNARE-dependent exocytosis by complexin II is unclear. In neuronal cells, it is widely known that complexin binds the SNARE complex immediately before exocytotic release. Therefore, it would be of great interest to determine the SNARE proteins that are regulated by complexin II in mast cells. In this study, we investigated the SNARE-binding partner of complexin in mast cells. To address this problem, we performed a glutathione S-transferase (GST) pulldown assay and an in vitro binding assay using recombinant SNARE proteins. Furthermore, we attempted to determine the
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region of complexin II that is responsible for binding to the SNARE complex in mast cells. 2. Materials and methods
total cell lysate of RBL-2H3 and incubated at 4 °C. After washing with His wash buffer [25 mM HEPES–KOH (pH 7.4), 1% Triton X100, 400 mM KCl, 2 mM b-mercaptoethanol, 10% glycerol, 50 mM imidazole], the eluate was subjected to SDS–PAGE and Western blot analysis.
2.1. Cell culture 2.6. Western blotting RBL-2H3 cells were cultured in Eagle’s minimal essential medium (Nissui, Tokyo, Japan) with 10% fetal bovine serum (GIBCO, Grand Island, NY) at 37 °C in a humidified atmosphere with 5% CO2. 2.2. Expression and purification of recombinant proteins Full-length rat syntaxin-3, VAMP-8, and SNAP-23 were expressed in Escherichia coli, and purified as described previously [32]. Full-length rat complexin II gene and rat complexin II deletion mutants were ligated into pGEX-4T 1 vector (Amersham Pharmacia Biotech, Uppsala, Sweden). The plasmids were then introduced into BL21 Star E. coli (Invitrogen, Carlsbad, CA). Transformed cells were grown in LB medium to an OD600 of 0.8, and protein expression was induced by 1 mM isopropyl b-D-L-thiogalactopyranoside (IPTG) treatment for 3 h at 37 °C. GST-complexin II was purified by MagneGST Protein Purification System (Promega, Madison, WI). In some experiments, GST was cleaved by thrombin. 2.3. Complexin II deletion mutants The primer pairs used to amplify complexin II lacking either the C-terminus (the region from residues 1–88) or the N-terminus (the region from residues 41–134), were 50 -GGATCCATGGACTTCGTC AT-30 (sense)/50 -CTCGAGCTGTTCCAGGGCTGCCTTCT-30 (anti-sense) and 50 -GGATCCCTGAGGCAGCAGGAGGAAGA-30 (sense)/50 -GCGGCC GCTTACTTCTTGAACATGTCCTGCA-30 (anti-sense), respectively. The obtained PCR fragments encoding complexin II lacking either the C-terminus or the N-terminus were subcloned into pGEX-4T 1 vector (Amersham Pharmacia Biotech). 2.4. Site-directed mutagenesis To obtain mutant complexin II(R59H), mutated complexin II in which CGT for Arg59 is substituted with CAT for His was prepared by the overlap extension method, using two pairs of oligonucleotides (50 -CCATGGACTTCGTCATGAAGCA-30 (sense)/50 -GGACCTTCTC ATGTTCCGCTTCCA-30 (anti-sense) and 50 -TGGAAGCGGAACATGAG AAGGTCC-30 (sense)/50 -GGATCCCTTCTTGAACATGTCCTGCA-30 (antisense), mutated codons are underlined. The obtained PCR fragment encoding the mutant complexin II(R59H) was subcloned into the NcoI–BamHI site of pQE60 (Qiagen, Hilden, Germany) and verified by DNA sequencing. 2.5. Pull-down assay A GST pull-down assay was performed using a GST Protein Interaction Pull-Down Kit (Pierce, Rockford, IL). In brief, cell lysate obtained from E. coli expressing GST-complexin II was mixed with glutathione agarose beads. GST-complexin II bound to glutathione beads was mixed with total cell lysate of RBL-2H3 and incubated at 4 °C. After washing with wash solution [a 1:1 mix of Tris-buffered saline (TBS) to ProFound Lysis Buffer (Pierce)], elution buffer was added and the eluate was subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis. For His pull-down assay, cell lysate obtained from E. coli expressing complexin II-His or complexin II(R59H)-His was mixed with Ni–NTA agarose beads (Qiagen). Complexin II-His or complexin II(R59H) bound to Ni–NTA agarose beads was mixed with
SDS–PAGE and Western blot analysis were performed as described previously [16]. Primary antibodies used are as follows: rabbit anti-SNAP-23 antibody (dilution 1:1000; Synaptic Systems, Göttingen, Germany), rabbit anti-syntaxin-3 antibody (1:1000; Synaptic Systems), rabbit anti-syntaxin-4 antibody (1:1000; Synaptic Systems), rabbit anti-VAMP-8 antibody (1:1000; Synaptic Systems), mouse anti-VAMP-7 antibody (1:100; Upstate Biotechnology, Lake Placid, NY), mouse anti-VAMP-2 antibody (1:2000; Synaptic Systems), mouse anti-Munc18 antibody (1:1000; BD Bioscience Pharmingen, San Diego, CA), and goat anti-synaptotagmin II (1:200; Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were detected by enhanced chemiluminescence (SuperSignal West Pico, Pierce) using a luminescence analyzer (LAS-3000 mini; FUJI FILM, Tokyo, Japan). 2.7. In vitro binding assay All reactions were performed in 400 ll binding buffer [50 mM Tris–HCl (pH 8.0), 100 mM KCl, 10% glycerol, 10 mM b-mercaptoethanol, and 0.8% b-octyl glucoside]. A sample of 3 lg GST-complexin II was immobilized on 20 ll of glutathione beads. SNARE proteins were added and the reaction mixture was maintained at 4 °C for 1 h. After washing with the binding buffer, sample buffer was added and eluted proteins were subjected to SDS–PAGE and Western blot analysis. 3. Results 3.1. Characterization of the SNARE complex that interacts with complexin II We tried to identify isoforms of SNAREs that interact with complexin II, and clarify the relationship between complexin II and other accessory SNARE proteins in mast cells. For this purpose, we obtained GST or GST-complexin II recombinant protein from transformed E. coli. These recombinant proteins were separated by SDS–PAGE and stained by Coomassie Brilliant Blue (CBB) to confirm their expression and purity. To determine the components of the SNARE complex that interact with complexin II in mast cells, a GST pull-down assay was carried out, and coprecipitated components were subjected to Western blot analysis (Fig. 1). We found that the SNARE complex that interacts with complexin II consisted of syntaxin-3, SNAP-23, VAMP-2, or VAMP-8. On the other hand, syntaxin-4 was not detected in the pull-down complex. We also investigated whether or not SNARE accessory proteins, such as Munc18-2 and synaptotagmin II, were present in this pull-down complex, but these proteins were not detected. This might be because Munc18 favors free syntaxin, and synaptotagmin competes with complexin for binding to the SNARE complex [30,33]. 3.2. Molecular interaction between complexin II and SNARE proteins We examined the binding affinity of complexin II to SNARE proteins to understand their binding. GST-SNAP-23, syntaxin-3-His, GST-VAMP-8, and GST-complexin II were expressed in E. coli and purified with affinity chromatography. Purified proteins were
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Fig. 1. SNARE proteins interacting with complexin II in mast cells. SNARE proteins that interact with complexin II were identified by GST pull-down assay. As a negative control, GST alone bound to glutathione beads was mixed with total cell lysates of RBL-2H3. Interacting proteins were detected by Western blot analysis. As a positive control, 3 lg total cell lysates of RBL-2H3 were loaded (n = 5).
electrophoresed by SDS–PAGE and stained with CBB (Fig. 2A). The purified GST-complexin II bound to glutathione beads was mixed with recombinant SNARE proteins. After washing with wash solution, the elution buffer was added and bound materials were subjected to SDS–PAGE and Western blot analysis (Fig. 2B). VAMP-8 and syntaxin-3 was able to bind to GST-complexin II by themselves, but SNAP-23 could not. Syntaxin-3 was able to bind to complexin II as well by itself. The binding affinity of complexin II with syntaxin-3 was enhanced in the presence of VAMP-8 (0.13 lg/ 400 ll) and SNAP-23 (0.13 lg/400 ll) to syntaxin-3. These results suggest that complexin II binds to the SNARE complex with a higher affinity than VAMP-8 or syntaxin-3 alone.
3.3. Binding region of complexin II to the SNARE complex Next, we tried to determine the region of complexin II that is necessary for binding to the SNARE complex in mast cells. To address this problem, we obtained the 1–88 aa mutant that lacks the C-terminal amino acids 89–134, and the 41–134 aa mutant that lacks the N-terminal amino acids 1–40. We also prepared a point-mutated complexin II (R59H). Full-length and two deletion mutants were tagged with GST. Full-length and mutated complexin IIR59H were tagged with hexahistidine. Pull-down assay was carried out using these proteins. The pull-down complex was subjected to either SDS–PAGE followed by Western blot analysis (Fig. 3A, upper panel) or stained by CBB (Fig. 3A, lower panel). CBB staining revealed that approximately equal amounts of full-length and both deletion mutants were immobilized on glutathione beads. Both deletion mutants of complexin II bound to approximately equal amounts of SNARE proteins compared with full-length complexin II. On the other hand, complexin IIR59H reduced its binding to the SNARE complex (Fig. 3B). Probably the site chain of histidine is exhibited shorter than that of arginine that serves for a salt bridge with aspartic acid residue of VAMP-2 [34]. The aspartic acid of VAMP-2 corresponds to asparagine in VAMP-8, and this might explain the binding to complexin II with a lower affinity. These results demonstrate that the central a-helix region, especially Arg59, is responsible for binding to SNARE complex in mast cells.
Fig. 2. In vitro assay of interaction between SNARE proteins and complexin II. (A) GST-SNAP-23, syntaxin-3-His, GST-VAMP-8, and GST-complexin II were expressed in E. coli and purified. Samples were separated by SDS–PAGE and stained by CBB. (B) GST-complexin II (3 lg) bound to glutathione beads was mixed with one of SNAREs (SNAP-23, VAMP-8 and syntaxin-3) at indicated concentrations except for the lowest frame (SNAP-23 + Syntaxin-3 + VAMP-8) in which GST-complexin II was mixed with all of SNAREs (SNAP-23 (0.13 lg/400 ll) + Syntaxin-3 (indicated concentration) + VAMP-8 (0.13 lg/400 ll)). Pull-down samples were subjected to SDS–PAGE and Western blot analysis. When GST-complexin II were mixed with one of SNAREs, immunoreactivity was detected with a specific antibody for each SNAREs (upper three frames). When GST-complexin II were mixed with all of SNAREs, immunoreactivity was detected with a specific antibody for syntaxin-3 (the lowest frame). The binding affinity of complexin II with syntaxin-3 was enhanced in the presence of VAMP-8 (0.13 lg) and SNAP-23 (0.13 lg) to syntaxin-3 (n = 4).
4. Discussion We previously reported that complexin II is expressed in mast cells and the knockdown of complexin II inhibited degranulation. Moreover, we found that the knockdown of complexin II decreased Ca2+ sensitivity of the degranulation machinery [16]. However, it was not clear which SNARE proteins were regulated by complexin II. In this study, we investigated the SNARE complex that is regulated by complexin II in mast cells. The results of the pull-down assay and the in vitro binding assay suggest that the SNARE complex containing syntaxin-3 is regulated by complexin II in mast cells. As shown in Fig. 2B, complexin II binds to syntaxin-3, and this binding was enhanced by addition of SNAP-23 and VAMP-8 (bottom panel). Although we have not direct evidence, the binding enhancement seems to be due to the formation of a stable SNARE complex comprised of SNAP-23, syntaxin-3, VAMP-8 and complexin II. If syntaxin-3 and VAMP-8 bind to complexin II at the same biding site of complexin II, binding of syntaxin-3 to complexin II would be decreased in the presence of VAMP-8. However, bound syntaxin-3 was increased in the presence of VAMP-8, sug-
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Fig. 3. Binding analysis of complexin II with SNAREs. Deletion mutants of complexin II (1–88 amino acids and 41–134 amino acids) were tagged with GST and immobilized on glutathione beads. As positive and negative controls, GSTcomplexin II and GST were used, respectively. These immobilized proteins were mixed with total cell lysates of RBL-2H3, and pull-down samples were subjected to SDS–PAGE and Western blot analysis (A, upper panel) or stained by CBB (A, lower panel) (n = 3). Complexin IIR59H was tagged with hexahistidine and immobilized Ni– NTA agarose beads. As a positive control, hexahistidine-tagged complexin II was used. These immobilized proteins were mixed with total cell lysates of RBL-2H3. As a negative control, Ni–NTA agarose beads alone was mixed with them. Pull-down samples were subjected to SDS–PAGE and Western blot analysis (B). Amounts of SNAP-23, syntaxin-3, VAMP-2 and VAMP-8 bound to complexin IIR59H decreased to 10.1 ± 2.6%, 35.3 ± 4.3%, 35.5 ± 5.2% and 58.9 ± 8.5% of control, respectively (n = 4).
gesting that the binding site is different each other and both syntaxin-3 and VAMP-8 are included in the complex. SNAP-23 did not bind to complexin II directly, but similar GST pull-down experiments using neural SNAREs have shown that SNAP-25 enhances binding of syntaxin-1 to complexin II by forming SNARE complex, while VAMP-2 has little effects [18,21]. Therefore, it is probable that the complex formed in the experiment shown in the bottom panel in Fig. 2B includes SNAP-23 as well as VAMP-8. Moreover, complexin II might interact with the SNARE complex across syntaxin and VAMP, since both syntaxin-3 and VAMP-8 can bind to complexin II by themselves. This configuration also agrees with the previous identification of crystal structure [34]. Pabst et al. investigated the association of complexin with the SNARE complex using different isoforms of syntaxin [35,36]. They showed that complexin binds to a SNARE complex comprising SNAP-25, VAMP-2, and syntaxin-3 as efficiently as it binds to complex com-
prised of SNAP-25, VAMP-2 and syntaxin-1. However, complexin did not bind to the SNARE complex containing syntaxin-4. Moreover, Reim et al. also reported that complexin II interacts with a SNARE complex comprising SNAP-25, VAMP-2, syntaxin-3 at retinal ribbon synapses [37]. These results suggest that complexin II binds to the SNARE complex through syntaxin-3 but not through syntaxin-4. As for VAMP, we found that complexin II interacted with SNARE complex containing VAMP-2 or -8 in mast cells. Recently, Malsam et al. reported that complexin I interacts with SNAP-25/syntaxin-1/VAMP-2 or -8 and affects the fusion of syntaxin-1/SNAP-25 liposome with VAMP-8 and VAMP-2 liposome [38]. Taking these results into account, complexin II seems to regulate the SNARE complex containing syntaxin-3 regardless of the subtype of VAMP. We also determined the region of complexin II that is necessary for binding to the SNARE complex in mast cells. Chen et al. reported that complexin I binds to the SNARE complex, which consists of syntaxin-1, SNAP-25, VAMP-2, through a central a-helix (the region between residue 48 and 70 of complexin I) and Arg59 in complexin contributes to crucial contacts with SNARE complex [34]. Moreover, point mutation of complexin (R59H) decreased SNARE complex binding and abolished its overexpression effects in neuron [39]. In mast cell, the central a-helix of complexin II is also important for binding to SNARE complex and complexin (R59H) may be able to use as a dominant negative form. In mast cells, Paumet et al. showed that syntaxin-4, but not syntaxin-3, immunoprecipitated with SNAP-23 [7]. They also found that overexpression of syntaxin-4 inhibited degranulation, whereas over expression of syntaxin-3 had not such effect [7]. Moreover, injection of anti-syntaxin-4 antibody caused inhibition of degranulation [12]. These results suggest that syntaxin-4 rather than syntaxin-3 is responsible for degranulation in mast cells. In contrast, we found that complexin II regulates degranulation in mast cells and binds to the SNARE complex through interaction with syntaxin-3 and not with syntaxin-4, suggesting the involvement of syntaxin-3 in degranulation. Several lines of evidence support this possibility. First, the cis-SNARE complex is formed from SNAP-23, syntaxin-3, and VAMP-7 [9]. Second, Munc18-2, one of the SNARE accessory proteins, regulates degranulation by interaction with syntaxin-3 in mast cells [14,15]. Third, in the SNARE reconstituted liposome fusion assay, syntaxin-3 as well as syntaxin-4 together with SNAP-23 and VAMP-8, is enough to induce membrane fusion [32]. As for isoforms of syntaxin involved in exocytosis in mast cells, both syntaxin-3 and -4 might be involved. One possible difference between them in the regulation of exocytosis is that syntaxin-3 dependent process is regulated by SNARE accessory proteins such as munc18-2 and complexin II, while syntaxin-4 dependent process is not [14–16]. Another point that might explain the involvement of two isoforms of syntaxin is that there are different types of secretory granules that utilize different syntaxin to fuse with the plasma membrane. Although mast cell is not a polar cell, there might exist distinct regions in the plasma membrane where either syntaxin-3 or -4 are enriched. In the case of MDCK (Madin-Darby canine kidney) cells, syntaxin-3 is localized in the apical plasma membrane and syntaxin-4 is localized in the basolateral plasma membrane. In these cells, syntaxin-3 and -4 are differently involved in membrane traffic to apical and basolateral membrane [40,41]. For isoforms of VAMP, VAMP-8 plays a major role in the degranulation since VAMP-2-deficient BMMC (bone marrow-derived mast cells) did not exhibit defects in degranulation [11]. Fig. 4 depicts the role of complexin II and SNAREs in the exocytotic membrane fusion. Complexin II interacts with SNARE complex through binding to syntaxin-3 and VAMP-8 with its central a-helical region. Syntaxin-4 also forms SNARE complex with SNAP-23 and VAMP-8, but complexin II does not seem to regulate this type of SNARE complex. As we reported previously, bind-
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Fig. 4. Schematic representation of regulation mechanism by complexin II and SNAREs in degranulation. SNAP-23 and syntaxin-3 are expressed on the plasma membrane. VAMP-8 is expressed on granular membranes. After stimulation, SNAP-23, syntaxin-3 and VAMP-8 assemble and form SNARE complex. Complexin II binds to this SNARE complex through syntaxin-3 and VAMP-8, stabilizing the complex. Sustained increase in intracellular Ca2+ concentration causes exocytotic membrane fusion. Complexin II may be released from the complex after membrane fusion as in neuronal cells. Although syntaxin-4 is also expressed and forms SNARE complex, complexin II does not bind to this type of SNARE complex.
ing of complexin II to SNARE complex alone does not trigger exocytotic membrane fusion [16]. It requires sustained increase in intracellular Ca2+ by Ca2+ influx from extracellular medium. In some types of secretory cells including mast cells, a membrane fusion between secretory granules is also observed [42,43]. It is believed that granule–granule fusion allows granules far from the plasma membrane to secrete intragranular contents. We previously reported that syntaxin-3 is found not only on the plasma membrane but also on the secretory granules in mast cells [8,15]. Therefore, syntaxin-3 on the secretory granule might be involved in both the fusion with the plasma membrane and granule–granule fusion to facilitate exocytotic release. Acknowledgments This work was supported in part by the Ministry of Education, Culture, Sports, and Technology of Japan (Grants 17049024 and 18370064 to N.H.), and Grant-in-Aid for Research in Nagoya City University to S.T. References [1] S.T. Holgate, The epidemic of allergy and asthma, Nature 402 (1999) B2–B4. [2] H. Turner, J.P. Kinet, Signaling through the high-affinity IgE receptor EceRI, Nature 402 (1999) B24–B30. [3] U. Blank, J. Rivera, The ins and outs of IgE-dependent mast-cell exocytosis, Trends Immunol. 25 (2004) 266–273. [4] T.H. Sollner, Regulated exocytosis and SNARE function, Mol. Membr. Biol. 20 (2003) 209–220. [5] Y.A. Chen, R.H. Scheller, SNARE-mediated membrane fusion, Nat. Rev. Mol. Cell Biol. 2 (2000) 98–106. [6] Z. Guo, C. Turner, D. Castle, Relocation of the t-SNARE SNAP-23 from lamellipodia-like cell surface projections regulates compound exocytosis in mast cells, Cell 94 (1998) 537–548. [7] F. Paumet, J. Le Mao, S. Martin, T. Galli, B. David, U. Blank, M. Roa, Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8 containing secretory compartment, J. Immunol. 164 (2000) 5850– 5857. [8] T. Hibi, N. Hirashima, M. Nakanishi, Rat basophilic leukemia cells express syntaxin-3 and VAMP-7 in granule membranes, Biochem. Biophys. Res. Commun. 271 (2000) 36–41. [9] N. Puri, M.J. Kruhlak, S.W. Whiteheart, P.A. Roche, Mast cell degranulation requires N-ethylmaleimide-sensitive factor-mediated SNARE disassembly, J. Immunol. 171 (2003) 5345–5352. [10] N. Tiwari, C.C. Wang, C. Brochetta, G. Ke, F. Vita, Z. Qi, J. Rivera, M.R. Soranzo, G. Zabucchi, W. Hong, U. Blank, VAMP-8 segregates mast cell-preformed mediator exocytosis from cytokine trafficking pathways, Blood 111 (2008) 3665–3674.
[11] N. Puri, P.A. Roche, Mast cells possess distinct secretory granule subsets whose exocytosis is regulated by different SNARE isoforms, PNAS 105 (2008) 2580– 2585. [12] L.E. Sander, S.P. Frank, S. Bolat, U. Blank, T. Galli, H. Bigalke, S.C. Bischoff, A. Lorentz, Vesicle associated membrane protein (VAMP)-7 and VAMP-8, but not VAMP-2 or VAMP-3, are required for activation-induced degranulation of mature human mast cells, Eur. J. Immunol. 38 (2008) 855–863. [13] N. Puri, P.A. Roche, Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis, Traffic 11 (2006) 1482–1494. [14] S. Martin-Verdeaux, I. Pombo, B. Iannascoli, M. Roa, N. Varin-Blank, J. Rivera, U. Blank, Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis, J. Cell Sci. 116 (2003) 325–334. [15] S. Tadokoro, T. Kurimoto, M. Nakanishi, N. Hirashima, Munc18-2 regulates exocytotic membrane fusion positively interacting with syntaxin-3 in RBL2H3 cells, Mol. Immunol. 44 (2007) 3427–3433. [16] S. Tadokoro, M. Nakanishi, N. Hirashima, Complexin II facilitates exocytotic release in mast cells by enhancing Ca2+ sensitivity of the fusion process, J. Cell Sci. 118 (2005) 2239–2246. [17] D. Baram, R. Adachi, O. Medalia, M. Tuvim, B.F. Dickey, Y.A. Mekori, R. SagiEisenberg, Synaptotagmin II negatively regulates Ca2+-triggered exocytosis of lysosomes in mast cells, J. Exp. Med. 189 (1999) 1649–1658. [18] H.T. McMahon, M. Missler, C. Li, T.C. Südhof, Complexins: cytosolic proteins that regulate SNAP receptor function, Cell 83 (1995) 111–119. [19] A. Abderrahmani, G. Niederhauser, V. Plaisance, M.E. Roehrich, V. Lenain, T. Coppola, R. Regazzi, G. Waeber, Complexin I regulates glucose-induced secretion in pancreatic beta-cells, J. Cell Sci. 117 (2004) 2239–2247. [20] C.M. Roggero, G.A. De Blas, H. Dai, C.N. Tomes, J. Rizo, L.S. Mayorga, Complexin/ synaptotagmin interplay controls acrosomal exocytosis, J. Biol. Chem. 282 (2007) 26335–26343. [21] H. Tokumaru, K. Umayahara, L.L. Pellegrini, T. Ishizuka, H. Saisu, H. Betz, G.J. Augustine, T. Abe, SNARE complex oligomerization by synaphin/complexin is essential for synaptic vesicle exocytosis, Cell 104 (2001) 421–432. [22] K. Reim, M. Mansour, F. Varoqueaux, H.T. McMahon, T.C. Südhof, N. Brose, C. Rosenmund, Complexins regulate a late step in Ca2+-dependent neurotransmitter release, Cell 104 (2001) 71–81. [23] H. Cai, K. Reim, F. Varoqueaux, S. Tapechum, K. Hill, J.B. Sorensen, N. Brose, R.H. Chow, Complexin II plays a positive role in Ca2+-triggered exocytosis by facilitating vesicle priming, PNAS 105 (2008) 19538–19543. [24] M. Xue, A. Stradomska, H. Chen, N. Brose, W. Zhang, C. Rosenmund, K. Reim, Complexins facilitate neurotransmitter release at excitatory and inhibitory synapses in mammalian central nervous system, PNAS 105 (2008) 7875–7880. [25] S. Ono, G. Baux, M. Sekiguchi, P. Fossier, N.F. Morel, I. Nihonmatsu, K. Hirata, T. Awaji, S. Takahashi, M. Takahashi, Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals, Eur. J. Neurosci. 10 (1998) 2143–2152. [26] M. Itakura, H. Misawa, M. Sekiguchi, S. Takahashi, M. Takahashi, Transfection analysis of functional roles of complexin I and II in the exocytosis of two different types of secretory vesicles, Biochem. Biophys. Res. Commun. 265 (1999) 691–696. [27] C.G. Giraudo, W.S. Eng, T.J. Melia, J.E. Rothman, A clamping mechanism involved in SNARE-dependent exocytosis, Science 313 (2006) 676–680. [28] J.R. Schaub, X. Lu, B. Doneske, Y.K. Shin, J.A. McNew, Hemifusion arrest by complexin is relieved by Ca2+-synaptotagmin I, Nat. Struct. Mol. Biol. 13 (2006) 748–750. [29] T. Melia Jr, Putting the clamps on membrane fusion: how complexin sets the stage for calcium-mediated exocytosis, FEBS Lett. 581 (2007) 2131–2139.
56
S. Tadokoro et al. / Cellular Immunology 261 (2010) 51–56
[30] J. Tang, A. Maximov, O.H. Shin, H. Dai, J. Rizo, T.C. Südhof, A complexin/ synaptotagmin 1 switch controls fast synaptic vesicle exocytosis, Cell 126 (2006) 1175–1187. [31] M. Xue, K. Reim, X. Chen, H.T. Chao, H. Deng, N. Brose, C. Rosenmund, Distinct domains of complexin I differentially regulate neurotransmitter release, Nat. Struct. Mol. Biol. 14 (2007) 949–958. [32] H. Sakiyama, S. Tadokoro, M. Nakanishi, N. Hirashima, Membrane fusion between liposomes containing SNARE proteins involved in mast cell exocytosis, Inflamm. Res. 58 (2009) 139–142. [33] Y. Hata, C.A. Slaughter, T.C. Südhof, Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin, Nature 366 (1993) 347–351. [34] X. Chen, D.R. Tomchick, E. Kovrigin, D. Araç, M. Machius, T.C. Südhof, J. Rizo, Three-dimensional structure of the complexin/SNARE complex, Neuron 33 (2002) 397–409. [35] S. Pabst, J.W. Hazzard, W. Antonin, T.C. Südhof, R. Jahn, J. Rizo, D. Fasshauer, Selective interaction of complexin with the neuronal SNARE complex, determination of the binding regions, J. Biol. Chem. 275 (2000) 19808–19818. [36] M. Pabst, D. Vainius, R. Langen, R. Jahn, D. Fasshauer, Rapid and selective binding to the synaptic SNARE complex suggests a modulatory role of complexions in neuroexocytosis, J. Biol. Chem. 277 (2000) 7838–7848.
[37] K. Reim, H. Wegmeyer, J.H. Brandstätter, M. Xue, C. Rosenmund, T. Dresbach, K. Hofmann, N. Brose, Structurally and functionally unique complexins at retinal ribbon synapses, J. Cell Biol. 169 (2005) 669–680. [38] J. Malsam, F. Seiler, Y. Schollmeier, P. Rusu, J.M. Krause, T.H. Sollner, The carboxy-terminal domain of complexin I stimulates liposome fusion, PNAS 106 (2009) 2001–2006. [39] D.A. Archer, M.E. Grraham, R.D. Burgoyne, Complexin regulates the closure of the fusion pore during regulated vesicle exocytosis, J. Biol. Chem. 24 (2002) 18249–18252. [40] F. Lafont, P. Verkade, T. Galli, C. Wimmer, D. Louvard, K. Simons, Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells, PNAS 96 (1999) 3734–3738. [41] S.H. Low, S.J. Chapin, C. Wimmer, S.W. Whiteheart, L.G. Kömüves, K.E. Mostov, T. Weimbs, The SNARE machinery is involved in apical plasma membrane trafficking in MDCK cells, J. Cell Biol. 141 (1998) 1503–1513. [42] Y. Kawasaki, T. Saitoh, T. Okabe, K. Kumakura, M. Ohara-Imaizumi, Visualization of exocytotic secretory processes of mast cell by fluorescence techniques, Biochem. Biophys. Acta 1067 (1991) 71–80. [43] T. Kishimoto, R. Kimura, T.T. Liu, T. Nemoto, Vacuolar sequential exocytosis of large dense-core vesicles in adrenal medulla, EMBO J. 25 (2006) 673–682.