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Of Human Neutrophils Exocytosis of Specific and Tertiary Granules Role of Vesicle-Associated Membrane Protein-2, Through Q-Soluble N -Ethylmaleimide-Sensitive Factor Attachment Protein Receptor/R-Soluble N This information is current as -Ethylmaleimide-Sensitive Factor Attachment of October 1, 2021. Protein Receptor Interaction, in the Exocytosis of Specific and Tertiary Granules of Human Neutrophils Faustino Mollinedo, Belén Martín-Martín, Jero Calafat, Downloaded from Svetlana M. Nabokina and Pedro A. Lazo J Immunol 2003; 170:1034-1042; ; doi: 10.4049/jimmunol.170.2.1034 http://www.jimmunol.org/content/170/2/1034 http://www.jimmunol.org/ References This article cites 52 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/170/2/1034.full#ref-list-1 Why The JI? Submit online. by guest on October 1, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Role of Vesicle-Associated Membrane Protein-2, Through Q-Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor/R-Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Interaction, in the Exocytosis of Specific and Tertiary Granules of Human Neutrophils1 Faustino Mollinedo,2* Bele´n Martı´n-Martı´n,* Jero Calafat,† Svetlana M. Nabokina,* and Pedro A. Lazo* We have examined the role of the R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) synapto- brevin-2/vesicle-associated membrane protein (VAMP)-2 in neutrophil exocytosis. VAMP-2, localized in the membranes of specific Downloaded from and gelatinase-containing tertiary granules in resting human neutrophils, resulted translocated to the cell surface following neutrophil activation under experimental conditions that induced exocytosis of specific and tertiary granules. VAMP-2 was also found on the external membrane region of granules docking to the plasma membrane in activated neutrophils. Specific Abs against VAMP-2 inhibited Ca2؉ and GTP-␥-S-induced exocytosis of CD66b-enriched specific and tertiary granules, but did not affect exocytosis of CD63-enriched azurophilic granules, in electropermeabilized neutrophils. Tetanus toxin disrupted VAMP-2 and inhibited exocytosis of tertiary and specific granules. Activation of neutrophils led to the interaction of VAMP-2 with the plasma http://www.jimmunol.org/ membrane Q-SNARE syntaxin 4, and anti-syntaxin 4 Abs inhibited exocytosis of specific and tertiary granules in electroperme- abilized neutrophils. Immunoelectron microscopy showed syntaxin 4 on the plasma membrane contacting with docked granules in activated neutrophils. These data indicate that VAMP-2 mediates exocytosis of specific and tertiary granules, and that Q- SNARE/R-SNARE complexes containing VAMP-2 and syntaxin 4 are involved in neutrophil exocytosis. The Journal of Immu- nology, 2003, 170: 1034–1042. xocytosis plays an important role in neutrophil physiol- in a deregulated way. Specific and tertiary granules constitute a ogy, and secretion of readily mobilizable granules seems reservoir of plasma membrane proteins that are translocated to the by guest on October 1, 2021 E to regulate early neutrophil responses, including adhe- cell surface upon neutrophil activation, and contain a wide array of sion, extravasation, and the onset of respiratory burst (1, 2). Hu- proteins involved in the adhesion and extravasation of human neu- man neutrophils contain four major different types of granules, trophils as well as enzymes implicated in the generation of soluble namely: azurophilic or primary granules, specific or secondary mediators of inflammation (1, 2, 5, 9–16). Thus, secretion of neu- granules, gelatinase-rich or tertiary granules, and alkaline phos- trophil granules must be tightly controlled to prevent deregulated phatase-rich granules, also named secretory vesicles (2–7). Azuro- release of harmful components or unwanted inflammatory events. philic granules are sluggishly mobilized upon neutrophil activa- The membrane fusion process requires a molecular mechanism tion, whereas specific, tertiary, and alkaline phosphatase-rich to juxtapose and fuse the two membranes involved, and a regula- granules are readily exocytosed following neutrophil activation (1, tory mechanism that confers specificity to a particular type of fu- 7–10). Azurophilic granules contain a large number of lytic en- sion. In neuronal tissue and other systems, fusion of a vesicle with zymes that can be detrimental to the surrounding tissue if secreted its target membrane is mainly mediated by a set of proteins col- lectively referred to as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs)3 (17). The SNARE hy- *Centro de Investigacio´n del Ca´ncer, Instituto de Biologı´a Molecular y Celular del pothesis, initially postulated for synaptic vesicle release in the neu- Ca´ncer, Consejo Superior de Investigaciones Cientificas-Universidad de Salamanca, ronal system, assumes that docking and fusion of vesicles with the † Salamanca, Spain; and Division of Cell Biology, The Netherlands Cancer Institute, plasma membrane is mediated by the specific interaction of vesicle Amsterdam, The Netherlands proteins, v-SNAREs, including synaptobrevins/vesicle-associated Received for publication June 17, 2002. Accepted for publication November 8, 2002. membrane proteins (VAMPs), with target plasma membrane-lo- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance cated proteins, t-SNAREs, including syntaxins, synaptosome-as- with 18 U.S.C. Section 1734 solely to indicate this fact. sociated protein (SNAP)-25, and SNAP-23 (25- and 23-kDa, re- 1 This work was supported by Grant FIS-01/1048 from the Fondo de Investigacio´n spectively) (18–20). Sanitaria, Grants SA-087/01 and CSI-1/01 from Junta de Castilla y Leo´n, and Grant The neuronal SNAREs VAMP-2, syntaxin 1A, and SNAP-25 SAF2000-0169 from the Ministerio de Ciencia y Tecnologı´a of Spain. B.M.-M. is the recipient of a predoctoral fellowship from the Ministerio de Educacio´n y Cultura of have been shown to assemble into a very stable ternary complex Spain. S.M.N. is the recipient of a Federation of European Biochemical Societies short-term postdoctoral fellowship. 2 Address correspondence and reprint requests to Dr. Faustino Mollinedo, Centro de 3 Abbreviations used in this paper: SNARE, soluble N-ethylmaleimide-sensitive fac- Investigacio´n del Ca´ncer, Instituto de Biologı´a Molecular y Celular del Ca´ncer, Con- tor attachment protein receptor; SNAP, synaptosome-associated protein; VAMP, ves- sejo Superior de Investigaciones Cientificas-Universidad de Salamanca, Campus icle-associated membrane protein; TeTx, tetanus toxin; PMN, polymorphonuclear Miguel de Unamuno, E-37007 Salamanca, Spain. E-mail address: [email protected] neutrophil. Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 1035 with a 1:1:1 stoichiometry, referred to as the core complex, that Neutrophil isolation and activation consists of a twisted four-helical bundle (VAMP-2 and syntaxin Neutrophils were isolated from fresh heparinized human peripheral blood 1A contributing with one ␣-helix each and SNAP-25 contributing as previously described (28, 33), resuspended at 3–5 ϫ 106 cells/ml in with two ␣-helices) with all the chains aligned in parallel (21). HEPES/glucose buffer (150 mM NaCl, 10 mM HEPES, 5 mM KCl, 1.2 mM MgCl2, 1.3 mM CaCl2, 5.5 mM glucose, pH 7.5), and incubated at Sequence comparison analyses have showed that all SNAREs ␮ ϳ 37°C for 10 min with 1 g/ml PMA. Release of granule markers following share a homologous domain of 60 aa, referred to as the SNARE PMA activation was determined as previously described (1, 6, 9). motif (22–24), that mediates the association of SNAREs into core complexes forming the above four-helical bundle. All known Subcellular fractionation SNARE motifs fall into two major subfamilies that contain either Resting neutrophils were resuspended in 50 mM Tris-HCl, pH 7.5 con- a conserved glutamine (Q-SNARE) or a conserved arginine taining 2 mM PMSF, and then disrupted by repeated freeze-thaw. Homog- (R-SNARE) at a central position, leading to a reclassification enates were centrifuged at 1,200 rpm in a Sorvall T 6000D centrifuge for 10 min, and the supernatant, representing the postnuclear extract, was of SNARE proteins into Q- and R-SNAREs (22). In the neuro- saved. After centrifugation of the postnuclear extract at 45,000 rpm in a nal SNARE complex, three glutamine residues from the Q-SNARE TLA rotor for 90 min at 4°C using an Optima TL Ultracentrifuge (Beck- motifs (one contributed by syntaxin 1A and two by SNAP-25) and man Instruments, Palo Alto, CA), supernatant (soluble fraction) and pellet one arginine residue from
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