title: 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.
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 S-transferase (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 LC-MS/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.
authors.affiliation: Department of Biochemistry, VP Chest Institute, University of Delhi, Delhi, India.
authors.forename: Prabhjot
authors.initials: P
authors.lastname: Singh
authors.forename: Prija
authors.initials: P
authors.lastname: Ponnan
authors.forename: Nivedita
authors.initials: N
authors.lastname: Priya
authors.forename: Tapesh K
authors.initials: TK
authors.lastname: Tyagi
authors.forename: Marco
authors.initials: M
authors.lastname: Gaspari
authors.forename: Shibu
authors.initials: S
authors.lastname: Krishnan
authors.forename: Giovanni
authors.initials: G
authors.lastname: Cuda
authors.forename: Paritosh
authors.initials: P
authors.lastname: Joshi
authors.forename: Jasvinder K
authors.initials: JK
authors.lastname: Gambhir
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Sharma
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Prasad
authors.forename: Luciano
authors.initials: L
authors.lastname: Saso
authors.forename: Ramesh C
authors.initials: RC
authors.lastname: Rastogi
authors.forename: Virinder S
authors.initials: VS
authors.lastname: Parmar
authors.forename: Hanumantharao G
authors.initials: HG
authors.lastname: Raj
mesh_terms: D000105:Acetyl Coenzyme A / Q000737:chemistry; D000123:Acetyltransferases / Q000737:chemistry* / Q000378:metabolism; D000215:Acylation; D000818:Animals; D037282:Calreticulin / Q000737:chemistry* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003374:Coumarins / Q000737:chemistry*; D005982:Glutathione Transferase / Q000737:chemistry*; D006189:Haemonchus / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012549:Schistosoma japonicum / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
chemical_list: D037282:Calreticulin; D003374:Coumarins; D011994:Recombinant Proteins; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D005982:Glutathione Transferase; D008239:Lysine