Autoacetylation of Purified Calreticulin Transacetylase Utilizing Acetoxycoumarin as the Acetyl Group Donor

Abstract

Our earlier reports documented that calreticulin, a multifunctional Ca2+-binding protein in endoplasmic reticulum lumen, possessed protein acetyltransferase function termed Calreticulin Transacetylase (CRTAase). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of receptor proteins by a model acetoxycoumarin, 7,8-Diacetoxy-4-methylcoumarin, was observed. Here, we have examined the autoacetylation property of CRTAase by immunoblotting and mass spectrometry. Ca2+ was found to inhibit CRTAase activity. The inhibition of both autoacetylation of CRTAase as well as acetylation of the receptor protein was apparent when Ca2+) was included in the reaction mixture as visualized by interaction with anti-acetyl lysine antibody. The acetylation of lysines residues: -48, -62, -64, -153, and -159 in N-domain and -206, -207, -209, and -238 in P-domain of CRTAase were located by high-performance liquid chromatography-electronspray ionization tandem mass spectrometry. Further, computer assisted protein structure modeling studies were undertaken to probe the effect of autoacetylation of CRTAase. Accordingly, the predicted CRTAase 3D model showed that all the loop regions of both N- and P-domain bear the acetylated lysines. Energy minimization of the acetylated residues revealed charge neutralization of lysines due to the N-epsilon-acetylation which may facilitate the interaction of CRTAase with the protein substrate and the subsequent transacetylase action.

Comparison of Protein Acetyltransferase Action of CRTAase with the Prototypes of HAT

“…During the course of CRTAase catalyzed acetylation of receptor proteins by DAMC, acetylation of CR was also observed [19–21]. Such autoacetylation property of CRTAase was ratified by immunoblotting with acetylated lysine antibody and mass spectrometry [19–21].…”

“…Such autoacetylation property of CRTAase was ratified by immunoblotting with acetylated lysine antibody and mass spectrometry [19–21]. The acetylation of lysine residues Lys-48, -62, -64, -153, and -159 in the N-domain and -206, -207, -209, and -238 in the P-domain of CRTAase, was identified by LC-MS/MS.…”

“…Computational structure modeling studies were performed to probe the effect of autoacetylation of CRTAase. Accordingly, the predicted CRTAase 3D model showed that all the loop regions of both N- and P-domain bear the acetylated lysines [21]. None of the lysine residues in the C-domain was observed to be covalently modified by acetylation.…”

“…These results revealed charge neutralization of acetylated lysine residues and breaking of intermolecular H-bond with neighboring acidic residues (such as aspartate and glutamate). Thus, the protein modification by way of acetylation of lysine residues could facilitate interaction of CR with other proteins [21]. The occurrence of acetylated CR residue (Lys-206) reported in certain cases of acute myeloid leukemia due to cytogenetic abnormalities showed evidence for the CR acetylation in vivo [60].…”

“…Our investigations on the NO-related biological actions such as vasorelaxation, inhibition of ADP-induced platelet aggregation in vitro and in vivo [13, 17], activation of iNOS, and inhibition of TNF- α induced IL-6 in human peripheral blood mono nuclear cells (PBMC) [18] lead us to formulate the cardinal role of PA in influencing the NOS-related protein functions by way of acetylation mediated by TAase. The identity of TAase with an endoplasmic reticulum luminal protein, calreticulin (CR), was evidenced by proteomic analysis such as N-terminal sequencing, immunoreactivity with anti-calreticulin antibody, and mass spectrometry [19–21], and this novel function of CR was termed calreticulin transacetylase (CRTAase). The acetyltransferases mediated reversible protein acetylation was originally reported as a key posttranslational modification identified in nuclear proteins [3, 5, 6, 22] and then extended to cytosolic and mitochondrial proteins [2, 4, 7] by the identification and functional characterization of HATs and histone deacetylases (HDACs) in cytosol and mitochondria [23–27].…”