## 4 Conclusions  
We found that glutamic acid, aspartic acid, and poly L-glutamic acid do not inhibit antigen-induced degranulation of RBL-2H3 cells, but acidic amino acid-containing dipeptides do demonstrate inhibitive activity without cytotoxicity. L-glutamic acid-containing dipeptides suppressed the degranulation of RBL-2H3 cells induced with a calcium ionophore A23187. We also found that His-Glu tends to suppress, but not significantly, the elevation in cytosolic calcium ion concentration upon antigen and A23187 stimulation, and inhibits microtubule reorganization in RBL-2H3 cells after antigen stimulation. The results of immunoblot analysis showed that His-Glu does not significantly affect the phosphorylation of Syk or PLCy, but impacts that of PI3K and Akt. Overall results suggest that there are three mechanisms underlying the inhibitory effect of acidic amino acid residue-containing dipeptides on the degranulation of RBL-2H3 cells: a chelating effect, inhibition of intracellular processes after calcium flux, and blocking microtubule reorganization. Figures 1B and 2B indicate that the chelating effect of acidic amino acid residue-containing dipeptides seems weak. Thus, we consider that acidic amino acid residue-containing dipeptides mainly inhibit intracellular processes after the increase in the cytosolic calcium ion concentration, such as the formation of the SNARE complex, and/or block the microtubule reorganization through downregulated phosphorylation of PI3K and Akt as shown in Figure 5. The exact mechanism of the degranulation-inhibitory activity of acidic amino acid residue-containing  
Figure 4: Effect of an acidic amino acid residue-containing dipeptide on signaling pathways leading to degranulation. IgE-sensitized RBL-2H3 cells were treated with 2.0 mM His-Glu and subsequently stimulated with antigen for 5 min to induce degranulation. Cell lysates were then prepared and used for immunoblotting. The phosphorylation levels of Syk, PLCy1, PLCy2, PI3K, and Akt were evaluated. The p-Syk, p-PLCy1, p-PLCy2, p-PI3K, and p-Akt represent phosphorylated Syk, phosphorylated PLCy1, phosphorylated PLCy2, phosphorylated PI3K, and phosphorylated Akt, respectively. The relative phosphorylation ratio indicates the ratio of the amount of phosphorylated protein to the amount of whole protein. A representative blot from two independent experiments is shown. Data are expressed as the mean \(\pm\) SEM. * \(p\) < 0.05 and ** \(p\) < 0.01 against control (antigen positive/His-Glu negative) by Dunnett’s test.  
dipeptides has not yet been elucidated, and further investigation is needed. Based on our data, L-glutamic acid-containing dipeptides can be used as food ingredients to attenuate allergic symptoms. Further studies are required to examine whether allergic symptoms can be ameliorated with L-glutamic acid-containing dipeptides in vivo.  
Conceptualization, T.S.; formal analysis, K.N., M.I. ([PERSON]) and T.S.; investigation, T.H., M.I. ([PERSON]), N.K., S.Y., A.O. and Y.T.; writing--original draft preparation, K.N.; writing--review and editing, K.N. and T.S.; supervision, K.N. and T.S.; funding acquisition, T.S. All authors have read and agreed to the published version of the manuscript.  
This research was funded by Grants-in-Aid for Scientific Research (KAKENHI Grant Number JP22K05512) from the Japan Society for the Promotion of Science.  
Not applicable.  
The data supporting the reported results are available on reasonable request from the corresponding author.  
The authors declare no conflicts of interest.