Abstract
In silico-docking studies from previous work have suggested that Lys-206 and lys-207 of calreticulin (CR) play a pivotal key role in its well-established transacetylation activity. To experimentally validate this prediction, we introduced three mutations at lysine residues of P-domain of CR: K → A, P (mut-1) (K -206, -209), P (mut-2) (K -206, -207) and P (mut-3) (K -207, -209) and analyzed their immunoreactivity and acetylation potential. The clones of wild-type P-domain (P (wt) ) and three mutated P-domain (P (mut-1), P (mut-2) and P (mut-3)) were expressed in pTrcHis C vector and the recombinant P (wt) , P (mut-1) , P (mut-2) and P (mut-3) proteins were purified by Ni-NTA affinity chromatography. Screening of the transacylase activity (TAase) by the Glutathione S Transferase (GST) assay revealed that the TAase activity was associated with the P (wt) and P (mut-1) while P (mut-2) and P (mut-3) did not show any activity. The immune-reactivity to anti-lysine antibody was also retained only by the P (mut-1) in which the Lys-207 was intact. Retention of the TAase activity and immunoreactivity of P (mut-1) with mutations introduced at Lys-206, Lys-209, while its loss with a mutation at Lys-207 residue indicated that lysine-207 of P-domain constitutes the active site residue controlling TAase activity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02659-1.