Cellular & Molecular Immunology (2018) 15, 353–366 & 2018 CSI and USTC. All rights reserved 2042-0226/18 $32.00 www.nature.com/cmi

RESEARCH ARTICLE

VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other

Laura Gómez-Jaramillo1, Raquel Romero-García1, Gema Jiménez-Gómez1, Lisa Riegle2, Ana Belén Ramos-Amaya1, José Antonio Brieva1, Marie Kelly-Worden2 and Antonio Campos-Caro1

The production and secretion of antibodies by human plasma cells (PCs) are two essential processes of humoral immunity. The secretion process relies on a group of known as soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs), which are located in the plasma membrane (t-SNAREs) and in the antibody-carrying vesicle membrane (v-SNARE), and mediate the fusion of both membranes. We have previously shown that SNAP23 and STX4 are the t-SNAREs responsible for antibody secretion. Here, using human PCs and antibody-secreting cell lines, we studied and characterized the expression and subcellular distribution of vesicle associated (VAMP) isoforms, demonstrating that all isoforms (with the exception of VAMP1) are expressed by the referenced cells. Furthermore, the functional role in antibody secretion of each expressed VAMP isoform was tested using siRNA. Our results show that VAMP2 may be the v-SNARE involved in vesicular antibody release. To further support this conclusion, we used tetanus toxin light chain to cleave VAMP2, conducted experiments to verify co-localization of VAMP2 in antibody-carrying vesicles, and demonstrated the coimmunoprecipitation of VAMP2 with STX4 and SNAP23 and the in situ interaction of VAMP2 with STX4. Taken together, these findings implicate VAMP2 as the main VAMP isoform functionally involved in antibody secretion. Cellular & Molecular Immunology (2018) 15, 353–366; doi:10.1038/cmi.2016.46; published online 12 September 2016

Keywords: exocytosis; human; immunoglobulin; plasma cells; VAMP proteins

INTRODUCTION membrane fusion and, particularly, the fusion between secretory After B-lymphocytes are exposed to antigen, these cells differ- vesicles and the plasma membrane for exocytosis must be entiate into the final stage of the B-cell lineage commitment, the specific to maintain organelle identity, cell function and viability, plasma cells (PCs), which are known as the key effector elements and it requires a molecular mechanism to juxtapose and fuse the of the humoral immune response. PCs are regarded as specia- two membranes involved. This process is performed by a set of lized cells with the exclusive function of synthesizing and proteins called SNAREs (soluble N-ethyl-maleimide sensitive secreting immunoglobulins (Igs),1 although this assumption factor attachment protein receptors).8,9 SNARE proteins were has recently been challenged.2,3 PCs have a high secretory initially classified as v-SNAREs and t-SNAREs according to their capacity of up to several thousand antibody molecules per presence on either vesicle membranes or on the corresponding second.4 Therefore, they require extensive development of the target membranes, respectively. SNAREs also mediate homotypic secretory pathway cellular structures, including the endoplasmic membrane fusion, for example, the fusion of two similar vesicles, reticulum (ER), the polyribosomes and secretory vesicles, and and these proteins have more recently been classified as accordingly, they exhibit an increased cytoplasmic-to-nuclear R-SNAREs and Q-SNAREs according to the central amino acid ratio compared with the B-cell progenitors.5 contained in the zero layer.10 The v-SNAREs include proteins of Igs are confined to small carrier vesicles before secretion6 and the family (also called vesicle associated mem- are constitutively secreted from the cell into the extracellular brane proteins (VAMPs), from the vesicle-associated membrane space through the classical secretory pathway.7 The cell proteins), and the t-SNAREs include proteins of the

1Unidad de Investigación, Hospital Universitario Puerta del Mar, Cádiz, Spain and 2Department of Biology, Ball State University, Muncie, IN, USA Correspondence: Dr A Campos-Caro, Unidad de Investigación, Hospital Universitario Puerta del Mar, 9ª Planta, Avda. Ana de Viya, 21. CP, 11009 Cádiz, Spain. E-mail: [email protected] Received: 11 November 2015; Revised: 6 July 2016; Accepted: 6 July 2016 Synaptobrevins in human antibody-secreting cells L Gómez-Jaramillo et al 354

and SNAP-25 families. In general, and VAMPs monoclonal anti-VAMP2 (R&D systems, Minneapolis, MN, associate with the membrane via one transmembrane domain USA), rabbit polyclonal anti-VAMP2 (Abcam, Cambridge, (TMD),8,11 whereas SNAP-25 proteins are associated with UK), rabbit polyclonal anti-VAMP3 (Abcam), anti-VAMP4 membranes via several palmitate chains.12 There are numerous and anti-VAMP8 rabbit polyclonal antibodies (Synaptic Sys- isoforms of t-SNARE (Syntaxins and SNAP-25) and v-SNARE tems), rabbit polyclonal anti-VAMP5 (Biorbyt, Cambridge, UK (VAMPs),9,13 and the specific interactions among them could and Sigma-Aldrich, Madrid, Spain), mouse monoclonal anti- partially determine the different membrane compartments VAMP7 (Covalab, Cambridge, UK), goat anti-HA HRP (Horse- that are allowed to fuse within the cells.14 The SNARE radish peroxidase)-conjugated (GenScript, Piscataway, NJ, USA) proteins assemble into a core complex composed of a parallel and mouse monoclonal anti-β-actin (Abcam). HRP-conjugated four-helical coiled-coil bundle in which one helix belongs to one secondary antibodies were purchased from Sigma-Aldrich Syntaxin protein, another helix belongs to one VAMP protein (Madrid, Spain). For IF microscopy, rabbit anti-VAMP2 and another two helices belong to one SNAP-25 protein. The (Abcam), FITC-conjugated anti-human IgE (Biosource, Camar- interactions between SNARE proteins initially begin between illo, CA, USA), primary anti-human catalase, RAB9, LAMP1, their cytoplasmic N-terminal domains and proceed to the EEA1 antibodies (Cell Signaling Technologies, Danvers, MA, membrane-anchored C-terminal domains, bringing the oppos- USA) and Alexa-647 or Alexa-488 secondary anti-mouse or anti- ing membranes sufficiently close enough to facilitate fusion.15 rabbit antibodies (Invitrogen, Madrid, Spain) were used. For Toxins have been used to investigate the roles of SNAREs in ELISA, alkaline phosphatase-conjugated and unconjugated anti- cells. Many SNARE proteins are proteolytic substrates of body pairs against human IgE were obtained from Biosource. clostridial neurotoxins. Tetanus toxin and botulinum toxin are For the immunodot blots and for IF of plated vesicles, the examples of such toxins, and the cleavage of SNARE proteins selected primary antibodies were mouse anti-VAMP2 (Abcam) results in the inhibition of exocytosis; however, not all SNARE and goat anti-human IgG, IgM, and IgA (H+L) (Life Technol- isoforms are susceptible to these toxins.16 ogies, Carlsbad, CA, USA), and the secondary antibodies were It has been hypothesized that Ig secretion from PCs is anti-mouse Alexa-680 (Life Technologies), rabbit anti-goat Dylight 800 (Rockland, Gilbertsville, PA, USA) and goat anti- mediated by constant trafficking of vesicles containing Igs to mouse Alexa-488 and goat anti-rabbit tertiary antibody Alexa- the cell surface. On their arrival, the Igs are discharged by 568 (Life Technologies). exocytosis without further storage.17 As final mediators of the humoral immune response, secreted soluble antibodies usually Tissue samples and cell lines serve to protect the individual against infection. However, some The human cell lines U266 and IM9 were purchased from the antibodies and the immune complexes formed by them are also European Collection of Cell Cultures (ECACC, Salisbury, UK), directly or indirectly implicated in the pathology of auto- and the RPMI-1788 cell line was purchased from the Coriell immune diseases,18 atopic disease19 or gammopathy-associated 20 Institute for Medical Research Biorepository (Camden, NJ, renal diseases. Despite the importance of Ig secretion in USA). U266 and IM9 were maintained in RPMI-1640 medium disorders such as these, little research has been published on supplemented with 10% heat-inactivated fetal bovine serum the SNARE proteins involved in this process. (FBS), 2 mM glutamine, 100 IU/ml penicillin and 100 μg/ml fi In previous studies, SNAP23 has been identi ed and char- streptomycin at 37 °C in a humidified atmosphere containing acterized as the SNAP-25 family member, and STX, as the 5% CO2. The same culture conditions, excluding the glutamine syntaxin family member involved in Ig secretion by human addition, were used for the RPMI-1788 cell line. 21–23 PCs; however, the VAMP family member, that is, the other Multiple myeloma PC (MMPC) was purified from donated component necessary to form the SNARE complex, remains to bone marrow aspirate of diagnosed MM patients using mono- fi be identi ed. The aim of the present study was, therefore, to clonal anti-CD138-coated microbeads (Miltenyi-Biotec, Auburn, analyze which isoform/s of the VAMP protein/s is/are implicated CA, USA). Human tonsil PC (To-PC) was obtained and purified in Ig secretion by human PCs. Here, we describe the various as previously described.24 All human samples were obtained VAMP isoforms expressed by human PCs and antibody- from adult donors with written informed consent under the secreting cell lines detected by western blotting. In addition, supervision and approval of the Institutional Review Board we use immunofluorescence (IF) microscopy to elucidate the (Ethics Committee for Research at the Hospital Universitario subcellular distribution of each VAMP isoform and used other Puerta del Mar, Cadiz, Spain). integrated strategies, such as VAMP expression knockdown by small interfering RNA (siRNA), VAMP treatment with tetanus Western blotting toxin and dominant-negative assays, to demonstrate that Cells were washed twice with PBS buffer and centrifuged, and VAMP2 is involved in the secretion of Ig by human PCs. the cell pellets were re-suspended in lysis buffer (125 mM Tris– HCl, 2% SDS, 1 mM DTT, pH 6.8) containing a protease MATERIALS AND METHODS inhibitor cocktail (Sigma, Madrid, Spain). Following sonication, Antibodies the protein concentration was quantified using a micro-BCA The following antibodies against human proteins were pur- protein assay kit (Thermo Fisher Scientific, Waltham, MA, chased from the following suppliers: rabbit polyclonal anti- USA). Proteins (20 μg) were resolved by 15% SDS–PAGE and VAMP1 (Synaptic Systems, Göttingen, Germany), mouse transferred to an Immobilon-P membrane (Millipore, Billerica,

Cellular & Molecular Immunology Synaptobrevins in human antibody-secreting cells L Gómez-Jaramillo et al 355

MA, USA). Total protein lysates from human brain, serving as a osmotic samples). Approximately 40 μl of each sample was control, were obtained from Biochain (Newark, CA, USA). added to poly-d-lysine-coated coverslips and blocked with Membranes were blocked with 5% skim milk in buffer (10 mM non-fat-milk for 30 min. Either mouse anti-human VAMP2 Tris–HCl pH 8, 150 mM NaCl, 0.1% Tween-20) and incubated (Abcam) and/or goat anti-human IgG, IgM, and IgA (H+L) with the appropriate primary antibody overnight a 4 °C. After (Life Technologies) were added and allowed to incubate for 2 h three washes with TBS-Tween, the membranes were incubated at 4 °C. The antibody mixture was then removed, and the plates with HRP-conjugated secondary antibody for 1 h at rom were rinsed twice with PBS. Subsequently, a secondary antibody, temperature (RT) and then washed three more times with rabbit anti-goat Dylight 800 (Life Technologies), was added and, TBS-Tween. Bound antibodies were detected using the Immuno after three washing steps, a secondary goat anti-mouse Alexa Star Western C kit with a ChemiDoc XRS system (Bio-Rad, Fluor488 and a tertiary antibody, Alexa-568 goat anti-rabbit (Life Madrid, Spain). The blots were then re-probed with β-actin Technologies), were also added. In the controls, antibodies were antibody as a loading control. Bands were quantified using the added to the dishes, but they were not plated with vesicles. The Quantity One Bio-Rad program, and the signal ratio was images were analyzed by fluorescence microscopy, and the calculated for VAMP/β-actin. corresponding images were taken with a Canon Power Shot A650 IS camera (Canon Inc., Melville, NY, USA). Preparation of vesicles The cell lysate supernatant was centrifuged for 30 min at 45 000g. Coimmunoprecipitation (Co-IP) assays The pellet was scraped off, homogenized in 40 mM sucrose and U266 cells were solubilized in lysis buffer (50 mM Tris–HCl pH then added to a sucrose gradient (200–400 mM). After spinning 7,4, 150 mM NaCl, 2 mM EDTA, 1% (w/v) NP-40, 0,1% SDS for 2 h at 57 000g, a central band containing the vesicles was supplemented with protease inhibitor cocktail from Sigma) for removed with a syringe. This fraction was aliquoted and then 1 h at 4 °C in a rotating wheel. Following centrifugation at stored at − 80 °C until use. 12 000g for 10min at 4°C, the clarified supernatants were collected as total cell lysates. The samples were then immuno- Immunodot blot precipitated overnight at 4 °C, together with a pre-incubated Immunodot blots were performed on PVDF membranes to antibody attached to the anti-VAMP2-Dynabeads protein G detect VAMP2 and/or IgM. Briefly, 1 μl of vesicle samples were (Life Technologies) or an isotype mouse serum-protein G as a applied directly to PVDF membranes and then thoroughly dried negative control. The beads were subsequently collected with a and rinsed with methanol and PBS. Following a 1-h blocking magnetic stand, washed three times with lysis buffer and eluted step with Odyssey blocking buffer, product number 927-40100 with SDS–PAGE sample buffer. Thereafter, the referenced (LI-COR, Lincoln, NE, USA), a mouse anti-VAMP2 or a goat samples were boiled and subjected to western blotting (WB) anti-human IgM was added, diluted in the same blocking buffer, analysis with the STX4, SNAP23 and VAMP2 antibodies. and then incubated overnight at 4 °C. The membranes were then washed three times with PBS-triton X-100 (1%), and then once siRNA silencing assays more with PBS. The membranes were then incubated with For siRNA knockdown experiments using the U266 cell line, rabbit anti-goat Dylight 800 or goat anti-mouse Alexa fluor-680 siRNA On-TARGET plus SMART pool (Dharmacon, Lafayatte, for 1 h. Finally, after a washing cycle as indicated above, the CO, USA) references L-012498-00 for VAMP2, L-011934-00 for membranes were visualized using an Odyssey Infrared Imaging VAMP3, L-004241-00 for VAMP4, L-017684-00 for VAMP5, System (LI-COR). L-020864-00 for VAMP7 and L-013503-00 for VAMP8, were used to inhibit VAMP production, whereas D-001600-01 as IF microscopy siGLO RISC-free siRNA served as a negative control. Cells U266 cells were incubated on poly-lysine treated slides for 1 h at (2 × 106) were transfected by nucleofection with Amaxa Nucleo- 37 °C. They were then fixed in 4% (w/v) paraformaldehyde in fector II (Lonza, Barcelona, Spain) using 100 nM siRNA for each PBS for 15 min at 4 °C, washed three times with PBS and then condition and the program (X-005) recommended by the permeabilized with 0.1% Triton X-100 in PBS. After washing manufacturer. For all cases, the nucleofected cells were cultured three times with PBS, the cells were incubated in blocking for 48 h. After refreshing the culture media and normalizing the solution (5% normal FBS in PBS) for 1 h at RT to block number of cells for each particular condition, the cells were nonspecific antibody binding. The cells were then incubated cultured for an additional 24 h, and the cell pellets and super- with the appropriate primary antibody in blocking solution for natants were collected and analyzed as stated for each 2 h. After washing three times with PBS, the cells were incubated experiment. with the appropriate fluorescence-conjugated secondary anti- body for 1 h at RT in the dark. After three more washes with Constructs and expression of fusion proteins PBS, the protein localization in the cells was analyzed using a cDNA for producing full-length human wild-type VAMP2 laser confocal TCS-SL microscope system from Leica Micro- (wtVAMP2) and transmembrane domain deleted VAMP2 systems (Barcelona, Spain). Regarding vesicle IF, vesicle fraction (VAMP2-ΔTMD) proteins was generated by PCR from U266 samples from RPMI-1788 cells were diluted 1:10 in PBS and using oligonucleotide primers as follows (small letters indicate centrifuged for 30 min at 29 000g. The pellets were then diluted cloning sites, capital letters specific cDNA coding VAMP2); 1:1 using either dH20 (hypo-osmotic samples) or PBS (iso- 5′-cgatatcgtcgacaccATGTCGGCTACCGCTGCCAC-3′ as sense

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primer for both cDNAs, and 5′-ggatcctttaccggtGAGCTGAAG tonsil, PCs from MM patients, and two different antibody- TAAACTATG-3′ and 5′-ggatcctttaccggtTTGAGGTTTTTCCA secreting cell lines, U266 and IM9, were studied using WB and CCAG-3′ as antisense primer for wtVAMP2 and VAMP2- IF techniques. The expression of VAMP1 was absent in all PCs ΔTMD, respectively. The cDNAs were cloned in-frame to the tested (Figure 1). However, the other members, VAMP2, − 3, amino-terminus of the monomeric red fluorescent Ruby − 4, − 5, − 7and− 8, were detected, although differences in the protein25 and verified by DNA sequence analysis. The cDNA level of expression were observed among the tested PCs of the tetanus toxin light chain (TeNT-LC) (a kind gift from (Figure 1). Quantifications of the bands observed in Figure 1 Professor G. Schiavo, Institute of Neurology, University College and quantifications of the bands from other U266 WB experi- London) was amplified by PCR and sub-cloned into the pIRES2- ments (total n = 4) are included as Supplementary Table S1. As EGFP expression vector. U266 cells were transfected with 2 μgof expected, VAMP2 and VAMP3 were detected at higher levels in DNA plasmid for all constructs stated in the experiment human brain extracts compared with VAMP4, VAMP5, VAMP7 according to the manufacturer’s instructions using an Amaxa and VAMP8, which were expressed at very low levels (Figure 1, nucleofector. At 48 h after transfection, fluorescent cells were lane C). The most apparent differences among human Ig- isolated by fluorescence-activated cell sorting (FACS) and then secreting cells were detected for the IM9 cell line, which showed cultured for an additional 24 h. The cell pellets and supernatants reduced VAMP3 expression, and for the U266 cell line, which were then analyzed by microscopy, western blotting and ELISA. exhibited high VAMP7 expression compared with the other PC samples (Figure 1). Other significant reductions in expression Flow cytometry and FACS were observed for VAMP5 in MMPC samples. VAMP2, VAMP4 Transfection efficiencies were usually analyzed 48 h after electro- and VAMP8 displayed more uniform expression. All bands poration using a BD Biosciences FACSCalibur flow cytometer. detected in the PC samples had the same sizes as in the brain Data were analyzed using Cell Quest software (BD Biosciences, samples, excluding VAMP5, which presented two bands in IM9 Madrid, Spain). When isolation of transfected cells was required, cells and brain, and only one band in U266, To-PCs and a FACSAria sorter (BD Biosciences) was used. For the intracel- MMPCs samples. lular IgE flow cytometry analysis, post-transfected cells with the corresponding constructs were stained with anti-human IgE- FITC (Life Technologies) using the fixation and permeabilization IntraStain kit (Dako, Glostrup, Denmark) according to the manufacturer’s instruction. Transfected cells (Ruby positive cells), were analyzed using a FACSCalibur flow cytometer, and the mean fluorescence intensity (MFI) for intracellular IgE-FITC staining was determined.

ELISA Suspensions of siRNA-transfected cells or plasmid-transfected FACS-sorted cells were cultured in a 24-well plate using 5 × 105 cells per well or in a 96-well plate using 1 × 105 cells per well, respectively. After 24 h, cell-free supernatants were collected, and the level of IgE secretion was evaluated by sandwich ELISA in microtiter plates as previously reported.26 For quantifications of intracellular IgE, after the cell pellets were previously lysed in buffer (50 mM Tris–HCl pH = 8, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, protease inhibitors), ELISA were performed as described above.

Statistical analysis The results are generally expressed as the mean and SEM. Data were analyzed using the Mann–Whitney U test to determine Figure 1 Expression of VAMP proteins in human antibody-secreting significant differences between any two experimental groups, cells. Immunoblots revealing protein levels of vesicle associated except for Figure 3, in which the ANOVA test and Tukey membrane protein (VAMP) isoforms in U266 and IM9 cell lines post-hoc test were applied. P values o0.05 were considered and PCs isolated from human tonsils or from the bone marrow of significant. patients diagnosed with multiple myeloma are shown. Pellets corresponding to 3 × 105 cell per lane were resolved and analyzed β RESULTS with anti- -actin (Abcam,1:15 000), anti-VAMP1 (SySy 1:200), anti-VAMP2 (R&D, 1:1000), anti-VAMP3 (Abcam, 1:500), anti- Expression and cellular localization of VAMP proteins in VAMP4 (SySy 1:200), anti-VAMP5 (Biorbyt 1:50), anti-VAMP7 human antibody-secreting cells (Covalab 1:500) and anti-VAMP8 (SySy 1:200) antibodies. As a To determine, which of the seven VAMP protein members were positive control, 20 μg of total protein lysate from human brain was expressed in human PCs, samples of normal primary PCs from used. For band quantifications, see Supplementary Table S1.

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In general, a cytoplasmic vesicular localization, associated with knockdown VAMPs, U266 cells were nucleofected with specific the exocytic and/or the endocytic pathway, has been described siRNA, and the antibody secretion level was determined 3 days for VAMPs. However, the intracellular distribution of the VAMP after nucleofection. As shown in Figure 3a, efficient knockdown isoforms has been shown to differ between different cell types after siRNA treatment resulted in a significant reduction in the (for review ref. 13). Therefore, using IF techniques, we studied levels of each VAMP proteins compared with cells treated with a the cellular distribution of the VAMP isoforms expressed in the negative siRNA control. The degree of each VAMP knockdown U266 and IM9 cell lines and in MMPC from patients (Figure 2). ranged between 50 and 90% compared with the control treated The IF results showed that VAMP2, VAMP3, VAMP4 and cells. However, only the reduction of VAMP2 by siRNA VAMP8 presented a punctuated staining pattern predominantly (Figure 3b) had a statistically significant effect on antibody in the cytoplasm, indicating a vesicular localization. VAMP5 and secretion into the cultured medium. In contrast, siRNA- VAMP7 also presented cytoplasmic staining, although it was mediated knockdown of the other VAMP proteins did not show more intense, patched and perinuclearly localized. These results any significant effect. Because siRNA previously demonstrated a were similar in all cells tested and suggest that all VAMPs might reduction in Ig secretion,22 aspecific siRNA for STX4 was used function as potential v-SNAREs in the fusion of the antibody- as a positive control. The combination of two VAMP siRNA carrying vesicles with the plasma membrane. simultaneously, that is, for VAMP2+VAMP3 or VAMP7 +VAMP8, revealed no differences compared with the single Effects of VAMP protein reduction by siRNA on antibody siRNA (not shown). A similar phenomena, that is, no function- secretion ally significant effect after SNARE knockdown, has been To provide functional evidence for which VAMP proteins might previously described and attributed to a functional compensa- be implicated in the antibody secretion process, we next tion and/or protein redundancy.27 However, our data suggested examined the role played by each in this process by down- that VAMP2 might be implicated in the secretion of antibody by regulating each VAMP isoform using RNAi approaches. To human PCs.

Figure 2 Subcellular localization of vesicle associated membrane protein (VAMP) isoforms. U266, IM9 and MMPC cells were analyzed by indirect IF with anti-VAMP2 (R&D, 0.5 μg/μl), anti-VAMP3 (Abcam, 1:100), anti-VAMP4 (SySy 1:50), anti-VAMP5 (Sigma-Aldrich 1:100), anti-VAMP7 (Covalab 1:100) and anti-VAMP8 (SySy 1:50) antibodies. As secondary antibodies, an appropriated anti-mouse or an anti- rabbit Alexa-488 conjugated antibody was used. A representative image of the cellular distribution of each VAMP isoform is shown. Differential interference contrast merged images are shown. All images were obtained with a Leica TCS-SP confocal microscope.

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Figure 3 Effects of silencing of vesicle associated membrane proteins (VAMPs) on the secretion of IgE by PCs. U266 cells were transiently nucleofected with 100 μM siRNA against each VAMP isoform and small interfering RNA (siRNA) controls as described in ‘Material and methods’ section. Two days after transfection, an equal number of cells (5 × 105) for each condition were placed in a 24-well plate, cultured for 24 h and then the pellet and the supernatant were collected and analyzed by WB and ELISA, respectively. (a)The efficiency of VAMP knockdown was determined using assays examining the cell pellets by WB followed by specificbandquantification. The numbers under the bands indicate the mean ± s.e.m. (n = 4) of the remaining protein compared with the control siRNA used. (b)Quantification of IgE secretion from U266 cells. The IgE content in the supernatants from cultured VAMP siRNA-silenced cells after transfection with targeting siRNA or siControl was measured by ELISA. The relative level of IgE secretion for each targeting siRNA was compared with the non-targeting siRNA control. The number of independent experiments performed for each condition was n = 12 for siControl and VAMP2, n = 5 for VAMP5, n = 4 for STX4 and n = 9 for VAMP3, − 4, − 7and− 8. ANOVA and the Tukey post-hoc test were applied. Data represent the mean ± s.e.m., and the asterisk denotes *Po0.05.

Co-localization of Ig-carrying vesicles and VAMP2 protein antibodies compared with the negative control without primary To verify that Igs were contained in cytoplasmic membrane antibodies. When both primary antibodies were added together vesicles, cultures of RPMI-1788 cells, an IgM-secreting cell line, (Figure 4C panels d,e), co-localization was observed in the overlay were used. The presence of IgM in the supernatant and inside of image (panel f), indicating that the IgM-containing vesicles also the vesicle were tested in cell fraction by sucrose gradient contained VAMP2 in their membranes. An analysis of co- techniques and dot-blot assays. Both supernatants and the cell localization showed a Pearson’scorrelationcoefficient of vesicle-containing fraction were positive for IgM detection R = 0.9324 for signals obtained in panels d,e. This result was also (Figure 4A). Moreover, the vesicle fraction samples were spread corroborated using IF microscopy, detecting a partial co- onto poly-d-lysine coverslips and examined for the presence of localization, when U266 cells were examined simultaneously IgM by indirect IF, as indicated by Figure 4B. IF images were for IgE and VAMP2 (Figure 5, upper panels). To assess whether positive only when the vesicle fractions were lysed, showing that VAMP2 was restricted to antibody-containing vesicles, a double the Igs were contained in the lumen of the membrane vesicles. labeling assay was performed, combining the anti-VAMP2 To determine whether Ig-containing vesicles also contained antibody with antibodies directed to the lysosome marker VAMP2, purified vesicle fractions from RPMI-1788 cells were LAMP1, the early endosome marker EEA1, the late endosome laid onto poly-lysine-coated coverslips and then tested for co- marker RAB9 and the peroxisome maker catalase. The results labeling of IgM and VAMP2. As seen in Figure 4C, panels a–c, IF revealed no co-localization (Figure 5, merged panels). Consider- images displayed positive labeling for IgM or for VAMP2 ing these data together, we concluded that both Igs and VAMP2

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Figure 4 In vitro co-localization of VAMP2 with vesicles containing IgM in RPMI-1788 cells. (A) Supernatants or purified vesicle fractions from the IgM-secreting cells, RPMI-1788, were subjected to dot-blot assays. An IgM gradient (1-100 μg) was used as a positive control. (B) IgM is contained in vesicles. Osmotically unlysed (b) or lysed (c) vesicle fractions were laid on poly-lysine treated coverslips, probed with a goat anti-human IgM antibody and analyzed with a rabbit anti-goat Dyligth-800 antibody followed by a goat anti-rabbit tertiary antibody, Alexa-568, and visualized by IF microscopy. As a negative control (a), no primary antibody was added to the reaction. The results show that IgM could be only detected in the lysed vesicles. (C)Purified and lysed vesicle fractions were laid on poly-lysine coverslips and tested for co-labeling of VAMP2 and IgM. For VAMP2, (a) a mouse anti-human VAMP2 primary antibody and a goat anti-mouse Alexa Fluor488 conjugate as a secondary antibody was used. For IgM detection (b), goat anti-human IgM as a primary antibody and a rabbit anti-goat Dyligth-800 antibody as a secondary antibody followed by goat anti-rabbit Alexa-568 was used. For the negative control (c), no primary antibody was added to the reaction. For co-localization (d,e), a single coverslip preparation was incubated with both primary and secondary/tertiary antibody pairs described in a and b. An overlay image is shown (f), and arrows indicate some of the colocalizations. All images were acquired with a 10 × objective fluorescence microscope (Zeiss ISC 25). co-localized in vesicles, even in different cell lines producing (TeNT-LC),28,29 we decided to test whether antibody secretion different Ig isotypes. could be inhibited by expressing the TeNT-LC in U266 cells. To achieve this goal, TeNT-LC cDNA was cloned in a vector with Effect of TeNT-LC on Ig secretion in the U266 cell line an IRES-EGFP cassette and transfected in U266 cells. The Many of the SNARE proteins are targets for neurotoxins from transfection efficiency was approximately 50% (green cells Clostridium sp., and considering that VAMP2 and VAMP3 detected by cytometry). To avoid interference due to the other can be proteolyzed by the light chain of the Tetanus toxin 50% non-transfected cells, only TeNT-LC-expressing green cells

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Figure 5 Subcellular co-localization of vesicles containing Igs with VAMP2. (a) U266 cells were examined for co-localization of VAMP2 with IgE using IF confocal microscopy as described in ‘Materials and methods’ section. For VAMP2 detection, a mouse anti-human VAMP2 (1:100) as a primary antibody and a goat anti-mouse Alexa Fluor647 (1:500) as a secondary antibody were used. For IgE detection, direct IF with an anti-human IgE FITC-conjugated antibody (1:200) was used. A merged image with differential interference contrast (DIC) and a magnified image are shown. (b) U266 cells were examined for co-localization of VAMP2 with several subcellular markers. For LAMP1 (1:200), Rab9 (1:100) and catalase (1.800), rabbit primary antibodies and a goat anti-rabbit Alexa Fluor488-conjugated (1:500) as a secondary antibody were used. Overlapping images (merge) and DIC are shown in the right panels. Scale bars (10 μm) are included.

were isolated by cell sorting, and then the effect of the toxin on vector were used as a control for protein expression. β-actin was the level of antibody secretion was measured by ELISA. Figure 6a used as a loading control. The reduction observed during shows that secretion was significantly reduced to ~ 20%, a level secretion was not due to a decrease in cell viability similar to what previously observed using an siRNA specificfor (Supplementary Figure S1). To identify whether the simulta- VAMP2 (Figure 3b). TeNT-LC was fully functional and specific, neous knockdown of several VAMPs could produce greater as observed in Figure 6b, because both VAMP2 and VAMP3 inhibition, we decided to test the effect of VAMP4, VAMP5, proteins were largely, but not totally, removed, as observed in VAMP7 and VAMP8 siRNAs in previously TeNT-LC- the overexposed right panel. VAMP7, as a TeNT-LC-insensitive transfected U266 cells. As controls, siRNA for STX4, an empty protein, was not affected.30 Cells transfected with the empty vector, or the TeNT-LC vector were used. Figure 6c shows the Ig

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Figure 6 Effect on IgE secretion using Tetanus toxin light chain overexpression. Positive transfected U266 cells with an empty pIRES2- EGFP or with TeTN-LC-pIRES2-EGFP constructs were isolated by FAC sorting. (a) Similar numbers (5x105) of isolated cells were then cultured for 24 h, and the level of IgE secreted into the medium was measured by ELISA. Empty vector transfected cells were used as a control. Data represent the mean ± s.e.m. (n = 4), Mann–Whitney test, *Po0.05. (b) Cell pellets were used to confirm that TeTN-LC was functional and proteolyzed the VAMP2 and VAMP3 isoforms. VAMP7 and β-actin served as TeTN-LC-insensitive and loading controls, respectively. The right panel shows an overexposure of the left panel with arrows indicating residual VAMP2 and VAMP3 bands. (c)IgE content in the supernatants or in the cell pellet (d) from each cell transfected condition (empty vector = pIRES2-EGFP, STX4 = siRNA STX4, Tx = TeTN-LC-pIRES2-EGFP, and combinations of Tx+siRNA indicated) were measured by ELISA. Data represent the mean ± s.e.m. (n = 4), Mann–Whitney test, *Po0.05.

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secreted into the cultured media and Figure 6d, the Ig retained in shown in Figure 8a, we observed fluorescent spots in the cells the cells, both were measured by ELISA as is described in when two antibodies for VAMP2, raised in different species, Material and Methods. The results demonstrated that for all were used as a positive control (central panels). Only a few spots cases, the TeNT-LC produced a reduction of Ig secretion as were observed when a pair of VAMP2 and STX4 antibodies was observed in Figure 6a, but the inhibition of IgE secretion was no used, but they were localized in the plasma membrane (lower further reduced when any of the siRNA of VAMPs was panels). No signal was observed in negative control samples in additionally transfected. siRNA for STX4 alone displayed the which one of the primary antibodies was not included (upper expected inhibition but did not show any increase in the panels). The number of spots per cell for each condition is inhibition of Ig secretion when it was cotransfected with the shown in Figure 8b. Moreover, VAMP2 and STX4 interactions TeNT-LC. An inverse correlation of the amount of Ig retained in were also corroborated by Co-IP experiments (Figure 8c). the cells compared with that secreted was observed (Figure 6d). VAMP2 and STX4 were separately immunoprecipitated from U266 cell lysates, and after WB, both immunoprecipitates were Ig secretion is inhibited in U266 cells expressing VAMP2 probed for VAMP2, STX4 and SNAP23. As a control for IP, an lacking the TMD unspecific isotype antibody was used. As shown in Figure 8c, we VAMP proteins are anchored to the vesicle membrane by their found that, although not prominently, both STX4 and SNAP23 carboxyl-terminal TMD.8 To further support the involvement of were specifically coimmunoprecipitated with VAMP2. VAMP2 in the antibody secretion process and to alleviate the possible protein redundancy effect after siRNA or TeNT-LC DISCUSSION approaches, we decided to perform a dominant-negative It has been previously reported, that Ig secretion by PCs is assay.31,32 Thus, we constructed a wild-type VAMP2 (wtVAMP2) mediated by the two Q-SNARE proteins SNAP23 (ref. 21) and and VAMP2 protein lacking the TMD (VAMP2-ΔTMD), STX4.22,23 In the present work, we focused on the vesicle- bearing both monomer red fluorescent protein, Ruby, fused at associated R-SNARE proteins implicated in this process.33 their carboxyl-terminus. Ruby-only-expressing vector and Initially, we noticed that PCs express all VAMP family members, wtVAMP2-Ruby and VAMP2-ΔTMD-Ruby-fused protein- except VAMP1. All the other VAMP members were expressed in expressing vectors were transfected into U266 cells. Figure 7a all Ig-secreting cells tested, although with certain differences in shows that Ruby-only transfected cells, as expected, exhibited their expression levels, as has been recently reported for disperse fluorescence (left panel). The wtVAMP2-Ruby trans- adipocytes.34 The different expression levels of these VAMP fected cells gave rise to typical punctuate vesicular staining. In members do not seem to reflect a clear relationship with their contrast, VAMP2-ΔTMD-Ruby-transfected cells exhibited possible role in Ig secretion. Thus, VAMP2, − 3, − 4and− 8are markedly disperse and mislocalized fluorescence. The results of all regularly expressed in human PCs, but in the present work, the IgE secretion level obtained after the expression of these only VAMP2 could be determined to be directly involved in Ig constructs are shown in Figure 7b. VAMP2-ΔTMD expression secretion. Notably, MMPCs presented diminished VAMP5 provoked a significant decrease in IgE secretion compared with expression compared with the To-PCs and cell lines. Further Ruby alone or wtVAMP2-Ruby constructs, indicating that the work will be required to clarify this point. The cellular integrity of the VAMP2 protein is necessary to maintain normal distribution of expressed VAMPs by human PCs exhibited, in IgE secretion by PCs, and suggesting a competition of the deleted general, a pattern similar to that observed in other cell systems,35 isoform with the endogenous VAMP2 during its interaction with inasmuch as a given isoform can be localized in multiple the other SNARE partners. Again, this reduction in IgE secretion intracellular compartments in the same cell or the distribution was not due to a differential viability of the cells (Supplementary of a particular VAMP can be different in different cell types.13 Figure S2). If VAMP2 participates in the IgE secretion process, it VAMP3 has been associated with early and recycling is reasonable to assume that transfected cells expressing VAMP2- endosomes,36 and we observed light cytoplasmic vesicular ΔTMD constructs must show an intracellular accumulation of labeling with an increased presence close to the PC nucleus. IgE molecules. As shown in Figure 7c, IgE intracellular staining VAMP4, VAMP5 and VAMP7 also appeared mainly located next of transfected cells measured by the MFI was higher for VAMP2- to the cellular nucleus in PCs. These findings might be in ΔTMD cells compared with Ruby alone or wtVAMP2-Ruby concordance with previous observations in which VAMP4 was – constructs. associated with early endosomes to the trans-Golgi network36 38 or in which VAMP7 was associated with late endosome– Interaction between VAMP2 and STX4 in human PCs lysosomes and the trans-Golgi network,39 but not for VAMP5, We next investigated whether VAMP2 could mediate Igs which has been described in vesicles that mediate the transport secretion through its interaction with SNARE proteins located of GLUT4 to membranes in skeletal muscle cells.40 The at the plasma membrane. Considering that VAMP2 seems to be subcellular localization for VAMP4, VAMP5 and VAMP7 does responsible, at least in part, for Ig secretion by PCs, it was not indicate a possible relationship with the secretion of Igs. hypothesized that it had to be complexed with other SNAREs, VAMP8 was initially associated with endosomes,41 but it is now such as SNAP2321 and/or STX4,22,23 which are equally involved believed to participate in other trafficking routes that have in the antibody secretion process. To confirm this, we examined recently been described.42–44 VAMP8 is present in human PCs whether VAMP2 could interact in situ with STX4 using the and displays a uniform dotted labeling pattern in the cytoplasm. proximity ligation assay technique and confocal microscopy. As This cytoplasmic labeling suggests that VAMP8 may play a role

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Figure 7 Effect on IgE secretion using VAMP2 dominant-negative overexpression. Positive transfected U266 cells with an empty vector expressing mRUBY, VAMP2-mRUBY or VAMP2-ΔTMD-mRUBY were isolated by FAC sorting. (a) Representative confocal microscopy images of the subcellular distribution of the overexpressed proteins. (b) Similar numbers of isolated cells (5 × 105) where then cultured for 24 h, and the level of IgE secreted into the medium was measured by ELISA. Ruby transfected cells were used as a control. Data represent the mean ± s.e.m. (n = 7), Mann–Whitney test, *Po0.05. (c) Transfected cells were analyzed after intracellular staining for IgE. A representative histogram showing the mean fluorescence intensity (MFI) for each transfected construct is shown. Data represent the mean ± s.e.m. (n = 4), U-Mann–Whitney test, *Po0.05.

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in the transport of vesicles containing Igs to the plasma supporting the localization of VAMP2 in other vesicular membrane. However, our functional results did not support compartments such as early or late endosomes, lysosomes or this possibility in antibody secretion and led us to study in detail peroxisomes, or even in organelles such as mitochondria, the the cellular distribution of VAMP2, which displays a vesicular Golgi apparatus or the ER, was observed (not shown). pattern of distribution in the cytoplasm, in some manner, that Initially, we used siRNA assays to assess the functional role of differs from the one visualized for VAMP8. R-SNARE proteins in Ig secretion by PCs. The specific knock- The present study shows that in human PCs, VAMP2 is down of all VAMP proteins tested had no effect on Ig secretion confined to Ig-carrying vesicles. In addition, no evidence by PCs except for a small reduction observed with specific siRNA for VAMP2. The possible implication of VAMP2 in Ig secretion, as suggested by this latter result, was further analyzed using two different strategies. First, taking into account that TeNT-LC is known to proteolyze VAMP2 and VAMP3,28,29 its cDNA was transfected into U266 cells in which the TeNT-LC was overexpressed. The specific and almost total proteolysis of VAMP2 and VAMP3 proteins observed in our system again only produced a limited inhibition of Ig secretion, likely indicating that another VAMP member, or other proteins, could substitute for the functional role in Ig secretion when VAMP2 is removed. Furthermore, in contrast to the phenomenon observed in chromaffin cells, in which overexpression of TeNT-LC completely abolishes cate- cholamine secretion, an effect that could be rescued by expres- sing either VAMP2 or VAMP3,45 in the present study, VAMP3 was also proteolyzed and had no effect on the inhibition of Ig secretion. However, something similar to our results has been described for GLUT4 trafficking in adipocytes,46 for which the simultaneous knockout of VAMP2, VAMP3 and VAMP8 was necessary to interrupt GLUT4 externalization.47 Our results suggest that VAMP2 is implicated in human PC Ig secretion and that its role can be assumed by other VAMP proteins, but not VAMP3. In agreement with this interpretation, a recent study in the same cellular system has shown that VAMP3 knockdown by siRNA did not decrease the secretion of IgE by PCs.23 Moreover, the IM9 cell line has a high IgG secretion rate but presents a greatly diminished VAMP3 expression level

Figure 8 VAMP2 interactions with STX4 and SNAP23. (a)Duolink in situ proximity ligation assay was performed on U266 cells. Rabbit anti-human STX4 and mouse anti-human VAMP2 were used as primary antibodies to detect complexes. Omission of the primary antibodies served as a nonspecific negative control. As a positive control, rabbit and mouse anti-VAMP2 antibodies were used with the same preparation. Cells were visualized using differential interference contrast microscopy (left column), confocal fluorescence microscopy (IF, middle column), and the merge of both (right column). Arrows indicate spots located in plasma membranes. (b)Quantification of spots/cell obtained from 10 cells for each condition. U-Mann–Whitney test, *Po0.05. (c) Co-IP studies showing the interactions between VAMP2, SNAP23 and STX4. Total U266 cell lysates were immunoprecipitated with a nonspecific antibody isotype as a negative control, a specific antibody against VAMP2 or against STX4 bound to protein G-magnetic beads. Total lysates before (LB) and after (LA) immunoprecipitation and bound eluted (BE) samples were resolved by SDS–PAGE and analyzed by western blotting (WB) with the indicated antibodies.

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(Figure 1; Supplementary Table S1), suggesting that this VAMP from Fondos Europeos de Desarrollo Regional (FEDER), from the is not necessary for the secretion of Igs. Consejería de Innovación, Ciencia y Empresa-Junta de Andalucía The second strategy using truncation mutants was based on (P09-CTS-5445) to AC-C and an ASPiRE grant from Ball State the observation that the TMDs of SNARE proteins have an University (Indiana, USA) to MK-W. important role in membrane fusion.31,48 The dominant-negative approach obtained through expressing a soluble form of VAMP2 corroborated that this isoform was implicated in Ig secretion. These truncated mutants are known to act as selective inhibitors 1 Fagraeus A. Plasma cellular reaction and its relation to the formation of antibodies in vitro. Nature 1947; 159:499. of exocytosis, presumably by competing with endogenous 2 Dang VD, Hilgenberg E, Ries S, Shen P, Fillatreau S. 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J Immunol – We thank G Schiavo (Cancer Research UK London Research Institute) 2005; 175:66866693. 22 Gomez-Jaramillo L, Delgado-Perez L, Reales E, Mora-Lopez F, for providing the TeNT-LC cDNA. This work was supported by Mateos RM, Garcia-Poley A et al. Syntaxin-4 is implicated in the grants from the Spanish National Institute of Health ‘Carlos III’ secretion of antibodies by human plasma cells. JLeukocBiol2014; (FIS-PI1101091, FIS-PI071075) from Plan Nacional I+D+i 2008-2011 95:305–312.

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