Site-Directed Mutations of Calcium-Binding Sites Contribute to Reducing the Immunoreactivity of the EF-Hand Sarcoplasmic Calcium-Binding Protein in Scylla paramamosain.

In order to reduce the immunoreactivity of sarcoplasmic calcium-binding protein (SCP), site-directed mutations were used to replace key amino acids in the conformational epitopes and calcium-binding sites. The mutant SCPs (mSCPs) were expressed in Escherichia coli, and their immunoreactivities were analyzed using iELISA and basophil activation assays. Furthermore, the structural changes of mSCPs were determined from the circular dichroism spectra. The iELISA results showed that mSCPs could effectively inhibit the binding of wild-type SCP (wtSCP) to sensitive serum, with inhibition rates that reached 90%. Moreover, mSCPs could downregulate the expression levels of CD63 and CD203c on the basophil surface. Compared with wtSCP, the peak values were significantly changed, and the calcium binding ability was impaired, which explained the decline in immunoreactivities of the mSCPs. All of the data confirmed that this approach was effective in reducing the immunoreactivity of SCP and could be applied to other shellfish allergens.