Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation

Abstract

Vesicle-associated V-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans-SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

Keywords: atopy; degranulation; mast cell; membrane fusion; peptide; soluble N-ethylmaleimide-sensitive factor attachment protein receptor.

Figure 1

Design of mast cell soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) motif-patterned peptides. Peptides patterned after the α-helical regions of the SNARE motifs of SN23 (green), syntaxin 4 (red), and VAMPs (blue) were designed. The names of the peptides are shown in the motif diagram, and the residue number in the motif corresponding to each peptide is indicated above each diagram. The amino acid sequences of the synthetic peptides are shown in Table 1. The soluble domains of various VAMP proteins are also indicated. TMD, transmembrane domain.

Figure 2

Effect of synthetic peptides on the degranulation of RBL-2H3 mast cells. (A) RBL-2H3 mast cells grown on 24-well plates were treated with dinitrophenyl (DNP)-specific IgE and then exposed to the synthetic peptides (10 µM) together with streptolysin-O (SLO). After incubation for 30 min, the cells were stimulated with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) for degranulation. For comparison, protein transduction domain (PTD)-conjugated synthetic peptides were tested in the same manner as described above without SLO treatment. The soluble domains of vesicle-associated membrane protein 2 and 8 (Vp2S and Vp8S) were included in the experiment as positive controls. (B) Correlation between the results of degranulation from the synthetic peptides with SLO and the PTD-conjugated peptides without SLO. (C,D) The inhibitory effects of the synthetic peptides on the mast cell degranulation of distinct granule subsets. Mast cells sensitized with DNP-IgE were treated with PTD-conjugated peptides (C) or soluble proteins (D), followed by stimulation with DNP-BSA. Cell culture medium supernatants were assayed for the detection of degranulation. LY294002 was used as a positive control inhibitor of FcεRI-mediated degranulation in mast cells. All data are presented as the mean ± SD from three independent experiments. The statistical significance of differences was evaluated by analysis of variance (*p < 0.05).

Figure 3

Inhibitory effects of the synthetic peptides on SNARE-driven membrane fusion. (A) Lipid mixing assay of T-liposomes containing syntaxin 4 (Syn4) and SN23 together with V-liposomes prepared with Vp2 (black) or Vp8 (gray). The assay was monitored by measuring the dequenching of NBD fluorescence and the data were plotted as a function of time. The maximum NBD signal was obtained by adding 0.1% Triton X-100™. (B) The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion was completely blocked by the cytoplasmic domains of Vp2 (Vp2S, red) and Vp8 (Vp8S, green) at the concentration of 10 µM. (C) The inhibitory effects of synthetic peptides on lipid mixing using mast cell SNAREs including Vp2 (black) and Vp8 (gray). Each bar represents the mean ± SD of three independent experiments. The statistical significance of differences was evaluated by analysis of variance (*p < 0.05). (D) The curves represent the lipid mixing driven by SN23-Syn4-Vp2 or SN23-Syn4-Vp8 in the presence of the most effective peptides (Syn4N, Vp2N, and Vp8N).

Figure 4

The inhibitory effect of synthetic peptides on the mast cell degranulation of distinct granule subsets. (A) Inhibitory effect of the N-terminal-mimicking peptides on membrane fusion dependent on the state of partial zippering of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. The peptides were added before (black) or after the preincubation of T- and V-liposomes together at 4°C overnight (gray). Data are expressed as the mean ± SD. Significance: *p < 0.05. (B) The inhibitory effect of synthetic peptides on the degranulation of RBL-2H3 mast cells pretreated with A23187, which is an inducer of degranulation of mast cell without antigen-related FcεRI-crosslinking. After A23187 pretreatment, the mast cells were sensitized with DNP-IgE and then treated with protein transduction domain -conjugated peptides. The amounts of released granular components such as β-hexosaminidase and histamine were quantified by colorimetric biochemical assays. Data are expressed as the mean ± SD. Significance: *p < 0.05. (C) The extent of the formation of SNARE complexes in RBL-2H3 cells in the presence of inhibitory peptides was also examined by co-immunoprecipitation using an anti-SNAP-23 antibody.

Figure 5

Efficacy of N-terminal-mimicking peptides for treating the DNCB-induced atopic dermatitis-like skin lesions of NC/Nga mice. (A) Experimental schedule for application of the experimental drugs to the dermatitis-like skin lesions induced in the NC/Nga mice. Following an initial sensitization step with 1% DNCB twice weekly for 1 week, the mice were dorsally treated with 0.2% DNCB thrice weekly for 3 weeks. The NC/Nga mice were daily treated with peptide drugs for 4 weeks starting from week 9. (B) The skin inflammation in the NC/Nga mice was reduced by the synthetic peptides. Macroscopic appearance of mice treated with synthetic peptides (upper panels) and histopathologic evaluation after the repeated application of synthetic peptides to the back skin of NC/Nga mice stained with hematoxylin and eosin (middle) or toluidine blue (lower). Epidermal thickness (C), ear thickness (D), the number of mast cells (E), and the level of IgE (F) in serum were also evaluated. Dexamethasone (Dexa) was used as a positive control. Data are presented as the mean ± SD. *p < 0.05, ***p < 0.001 versus the control.