Autoacetylation of purified calreticulin transacetylase utilizing acetoxycoumarin as the acetyl group donor.
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.
Bansal
Seema
S
Department of Biochemistry, V. P. Chest Institute, University of Delhi, Delhi 1100 07, India.
Ponnan
Prija
P
Raj
Hanumantharao G
HG
Weintraub
Susan T
ST
Chopra
Madhu
M
Kumari
Ranju
R
Saluja
Daman
D
Kumar
Ajit
A
Tyagi
Tapesh K
TK
Singh
Prabhjot
P
Prasad
Ashok K
AK
Saso
Luciano
L
Rastogi
Ramesh C
RC
Parmar
Virinder S
VS
D000107:Acetylation / Q000187:drug effects; D000123:Acetyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000328:Adult; D000595:Amino Acid Sequence; D055162:Biocatalysis / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D003374:Coumarins / Q000378:metabolism*; D005260:Female; D006801:Humans; D006860:Hydrogen Bonding / Q000187:drug effects; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
C114369:7,8-diacetoxy-4-methylcoumarin; D003374:Coumarins; D010455:Peptides; D009251:NADPH-Ferrihemoprotein Reductase; D000123:Acetyltransferases; C539012:calreticulin transacetylase, human; D008239:Lysine; D002118:Calcium