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

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An Erratum to this article was published on 18 November 2008

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-ε-acetylation which may facilitate the interaction of CRTAase with the protein substrate and the subsequent transacetylase action.

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Acknowledgement

This work was supported by Department of Biotechnology, Government of India and Italian Ministry of University and Research, General Management of Strategies and Development of Internationalization of Scientific and Technological Research.

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Authors and Affiliations

  1. Department of Biochemistry, V. P. Chest Institute, University of Delhi, Delhi, 1100 07, India

    Seema Bansal, Prija Ponnan, Hanumantharao G. Raj, Ranju Kumari, Ajit Kumar, Tapesh K. Tyagi & Prabhjot Singh

  2. Department of Chemistry, University of Delhi, Delhi, 1100 07, India

    Prija Ponnan, Ashok K. Prasad, Ramesh C. Rastogi & Virinder S. Parmar

  3. Dr.B.R.Ambedkar Center for Biomedical Research, University of Delhi, Delhi 1100 07, Delhi, India

    Madhu Chopra & Daman Saluja

  4. Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA

    Susan T. Weintraub

  5. Department of Human Physiology and Pharmacology, “Vittorio Erspamer” Sapienza University of Rome, P.le Aldo Moro 5, 00185, Rome, Italy

    Luciano Saso

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  1. Seema Bansal
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  2. Prija Ponnan
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Correspondence to Hanumantharao G. Raj.

Additional information

An erratum to this article can be found at http://dx.doi.org/10.1007/s12010-008-8394-x

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Bansal, S., Ponnan, P., Raj, H.G. et al. Autoacetylation of Purified Calreticulin Transacetylase Utilizing Acetoxycoumarin as the Acetyl Group Donor. Appl Biochem Biotechnol 157, 285–298 (2009). https://doi.org/10.1007/s12010-008-8357-2

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