Polyphenols differentially inhibit degranulation of distinct subsets of vesicles in mast cells by specific interaction with granule-type-dependent SNARE complexes

Abstract

Anti-allergic effects of dietary polyphenols were extensively studied in numerous allergic disease models, but the molecular mechanisms of anti-allergic effects by polyphenols remain poorly understood. In the present study, we show that the release of granular cargo molecules, contained in distinct subsets of granules of mast cells, is specifically mediated by two sets of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, and that various polyphenols differentially inhibit the formation of those SNARE complexes. Expression analysis of RBL-2H3 cells for 11 SNARE genes and a lipid mixing assay of 24 possible combinations of reconstituted SNAREs indicated that the only two active SNARE complexes involved in mast cell degranulation are Syn (syntaxin) 4/SNAP (23 kDa synaptosome-associated protein)-23/VAMP (vesicle-associated membrane protein) 2 and Syn4/SNAP-23/VAMP8. Various polyphenols selectively or commonly interfered with ternary complex formation of these two SNARE complexes, thereby stopping membrane fusion between granules and plasma membrane. This led to the differential effect of polyphenols on degranulation of three distinct subsets of granules. These results suggest the possibility that formation of a variety of SNARE complexes in numerous cell types is controlled by polyphenols which, in turn, might regulate corresponding membrane trafficking.

Figure 1. Identification of active SNARE complexes in the mast cells

(a) mRNA expression of various SNAREs was analysed by reverse transcription–PCR. VAMP8 was repeatedly analysed with another primer set covering only the soluble SNARE motif (denoted by VAMP8S). (b) The lipid mixing ability of various SNARE complexes. Fusion traces for SNAP-25-containg complexes are shown in Supplementary Figure S3 at http://www.biochemj.org/bj/450/bj4500537add.htm. (c) Summary of SNARE protein expression in mast cell and lipid mixing ability of SNARE complexes.

Figure 2. Differential inhibitory effects of polyphenols DL, RT, GA and MR on the membrane fusion driven by different SNARE complexes

Percentage of maximal fusion was plotted as a function of time in the presence or absence of polyphenols. Polyphenols were treated at 10 µM concentration. The maximum fluorescence intensity was obtained by adding 0.1% Triton X-100.

Figure 3. Differential binding of polyphenols to different SNARE complexes observed by bathochromic shift

UV absorption spectra of polyphenols before and after mixing with soluble SNARE proteins were overlapped. (a–c) Bathochromic shifts of DL, QT and FS are induced only by Syn4/SNAP-23/VAMP8 complex, but not by Syn4/SNAP-23/VAMP2 complex; (d–e) bathochromic shifts of CY and RT are induced by only Syn4/SNAP-23/VAMP2 complex; (f) KP does not show any bathochromic shift with SNARE proteins; (g and h) bathochromic shifts of MR and LT by both complexes; (i) scheme of panel array.

Figure 4. Correlation between mast cell degranulation and SNARE-driven membrane fusion affected by polyphenols

(a–d) Scatter plots for inhibition of Syn4/SNAP-23/VAMP2-or Syn4/SNAP-23/VAMP8-driven fusion compared with β-hexosaminidase or histamine release. Dense areas are shown enlarged in Supplementary Figure S6 at http://www.biochemj.org/bj/450/bj4500537add.htm for better resolution between polyphenolic compounds. R² and slope (S) shown in the inset are the values considering all polyphenols. A linear relationship between the inhibition of SNARE-driven fusion and inhibition of degranulation by some polyphenols is highlighted in the shaded area. Polyphenols with relatively small inhibitory effect (<30%) are included in the box. (e) Inhibition of histamine release by polyphenols was extrapolated by using the simultaneous equation H=ax + by, where H is the inhibition percentage of histamine release by a polyphenol, × is the inhibition percentage of VAMP2-driven fusion by the polyphenol, y is the inhibition percentage of VAMP8-driven fusion by the polyphenol, and a and b are extrapolated constants.

Figure 5. Co-immunoprecipitation assay of different SNARE complexes in mast cells

Western blot analysis was performed using anti-SNAP-23 antibody after immunoprecipitating with several SNARE-specific antibodies. (b) Western blot analysis was performed using anti-Syn4 antibody after immunoprecipitating with several SNARE-specific antibodies. (c and d) Correlation between SNARE-mediated membrane fusion (lipid mixing assay) and SNARE complex formation in RBL-2H3 cells (co-immunoprecipitation). (e) Effect of polyphenols on the mRNA expression of SNARE genes in RBL-2H3 cells.