Protein Acyltransferase Function of Purified Calreticulin: The Exclusive Role of P-Domain in Mediating Protein Acylation Utilizing Acyloxycoumarins and Acetyl CoA as the Acyl Group Donors

Prabhjot      Singh; Prija      Ponnan; Nivedita      Priya; Tapesh      K. Tyagi; Marco      Gaspari; Shibu      Krishnan; Giovanni      Cuda; Paritosh      Joshi; Jasvinder      K. Gambhir; Sunil      K. Sharma; Ashok      K. Prasad; Luciano      Saso; Ramesh      C. Rastogi; Virinder      S. Parmar; Hanumantharao      G. Raj

Abstract

The distinct biochemical function of endoplasmic reticulum (ER) protein Calreticulin (CR) catalyzing the transfer of acyl group from acyloxycoumarin to a receptor protein was termed calreticulin transacylase (CRTAase). The present study, unlike the previous reports of others utilizing CR-deficient cells alone, dealt with the recombinant CR domains of Heamonchus contortus (rhCRTAase) in order to examine their CRTAase activity. P-domain of rhCR unlike N- and C-domains was found to be endowed with CRTAase function. We have also observed for the first time acetyl CoA, as a substrate for rhCRTAase/P-domain mediated acetylation of recombinant Schistosoma japonicum glutathione Stransferase (rGST). rhCRTAase/P-domain were also found to undergo autoacylation by acyloxycoumarins. Also, the isolated autoacylated rhCRTAase/P-domain in non-denatured form alone exhibited the ability to transfer acyl group to rGST indicating the stable intermediate nature of acylated CR. P-domain catalyzed acetylation of rGST by 7,8-Diacetoxy-4- methylcoumarin or acetyl CoA resulted in the modification of several lysine residues in common was evidenced by LCMS/ MS analysis. The putative site of the binding of acyloxycoumarins with CR was predicted by computational blind docking studies. The results showed the involvement of two lysine residues Lys-173 and Lys-174 present in P-domain for binding acyloxycoumarins and acetyl CoA thus highlighting that the active site for the CRTAase activity would reside in the P-domain of CR. Certain ER proteins are known to undergo acetylation under the physiological conditions involving acetyl CoA. These results demonstrating CRTAase mediated protein acetylation by acetyl CoA may hint at CR as the possible protein acetyltransferase of the ER lumen.

Keywords: Acetyl CoA, acyloxycoumarin, calreticulin, Haemonchus contortus, P-domain, CR, DAMC, GST, GSH, hCR, rhCRTAase/P-domain, Nanoscale LC-MS/MS, TFIIB, CYR, ER lumen proteinAcetyl CoA, acyloxycoumarin, calreticulin, Haemonchus contortus, P-domain, CR, DAMC, GST, GSH, hCR, rhCRTAase/P-domain, Nanoscale LC-MS/MS, TFIIB, CYR, ER lumen protein