1 3 Syntaxin-4 is essential for IgE secretion by plasma cells q 4 5 6 Arman Rahman a,1 Q1 , Joseph DeCourcey a,1 , Nadia Ben Larbi a , Sinéad T. Loughran b , 7 Dermot Walls b , Christine E. Loscher a,⇑ 8 a Immunomodulation Group, School of Biotechnology, Dublin City University, Ireland 9 b School of Biotechnology and National Centre for Sensor Research, Dublin City University, Ireland 10 11 13 article info 14 Article history: 15 Received 6 September 2013 16 Available online xxxx 17 Keywords: 18 Syntaxin-4 19 VAMP3 20 Plasma cells 21 IgE secretion 22 SNARE 23 Multiple myeloma 24 25 abstract 26 The humoral immune system provides a crucial first defense against the invasion of microbial pathogens 27 via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally 28 differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immune 29 response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly 30 understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor 31 (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin- 32 4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma mem- 33 brane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in 34 the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of 35 IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells 36 suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syn- 37 taxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mech- 38 anisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of 39 this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and 40 autoimmune disease where over-production of Ig leads to severe pathology in patients. 41 Ó 2013 The Authors. Published by Elsevier Inc. All rights reserved. 42 43 44 45 1. Introduction 46 Plasma cells are terminally differentiated, non-dividing final 47 effectors of the humoral immune response. As the sole professional 48 antibody producing cells in the body, they act as cellular factories 49 rapidly secreting vast amounts of immunoglobulin (Ig) [1]. Plasma 50 cells can be long lived, surviving for a number of years, and their 51 potential to produce large amounts of Ig necessary for the 52 clearance pathogens means that they are tightly regulated in the 53 immune response [2]. IgE, a variant of Ig produced in terminally 54 differentiated plasma cells, is not stored in the cell or packaged 55 in granules but is transported from the golgi via constitutive 56 pathways directly to the plasma membrane where it is secreted 57 [3]. The transportation of protein loaded vesicles from the golgi 58 to the plasma membrane and its subsequent fusion with the plas- 59 ma membrane is regulated by a conserved family of membrane 60 associated trafficking proteins called SNAREs (N-ethylmaleimide- 61 sensitive factor attachment protein receptor) [4]. 62 SNARE proteins share a highly conserved alpha helical SNARE 63 motif and mediate the docking and fusion of vesicular carriers crit- 64 ical for secretory immune functions [5]. Depending on the central 65 functional residue amino acid in the conserved SNARE motif, the 66 family is further divided into R-SNAREs (arginine) or Q-SNAREs 67 (glutamine) with Q-SNAREs being further classified into Qa, Qb 68 and Qc [6]. One SNARE motif from each group is required to form 69 a fully functional four-helical SNARE complex with at least one 70 SNARE present on the opposing membrane. The SNAREs are all 71 anchored at the C-terminus allowing the SNARE helices to ‘‘zipper 72 together’’ forming a stable complex that provides enough free 73 energy to overcome opposing membrane forces and allow the 74 membranes to fuse, resulting in the contents of the vesicle being 75 secreted from the cell [7]. 76 Syntaxin 4 in particular is a Qa SNARE that has been shown to 77 play an important role in regulated secretion on the plasma mem- 78 brane of neutrophils, macrophage and eosinophils (reviewed in 79 [5]). Despite a number of studies describing the secretion pathway 0006-291X/$ - see front matter Ó 2013 The Authors. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.09.058 Abbreviations: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; VAMP, vesicle-associate membrane protein; TNF, tumour necrosis factor; Ig, immunoglobulin; FBS, fetal bovine serum; PBS, phosphate buffered saline; siRNA, small interfering RNA. q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which per- mits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⇑ Corresponding author. Address: Immunomodulation Group, School of Biotech- nology, Dublin City University, Dublin 9, Ireland. E-mail address: christine.loscher@dcu.ie (C.E. Loscher). 1 These authors contributed equally to this work. Biochemical and Biophysical Research Communications xxx (2013) xxx–xxx Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc YBBRC 30889 No. of Pages 5, Model 5G 20 September 2013 Please cite this article in press as: A. Rahman et al., Syntaxin-4 is essential for IgE secretion by plasma cells, Biochem. Biophys. Res. Commun. (2013), http:// dx.doi.org/10.1016/j.bbrc.2013.09.058