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Characterization of protein transacetylase from human placenta as a signaling molecule calreticulin using polyphenolic peracetates as the acetyl group donors

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Abstract

We have earlier shown that a unique membrane-bound enzyme mediates the transfer of acetyl group(s) from polyphenolic peracetates (PA) to functional proteins, which was termed acetoxy drug: protein transacetylase (TAase) because it acted upon several classes of PA. Here, we report the purification of TAase from human placentral microsomes to homogeneity with molecular mass of 60 kDa, exhibiting varying degrees of specificity to several classes of PA confirming the structure-activity relationship for the microsome-bound TAase. The TAase catalyzed protein acetylation by a model acetoxy drug, 7,8-diacetoxy-4-methyl coumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with anti-acetyl lysine antibody. TAase activity was severely inhibited in calcium-aggregated microsomes as well as when Ca2+ was added to purified TAase, suggesting that TAase could be a calcium binding protein. Furthermore, the N-terminal sequence analysis of purified TAase (EPAVYFKEQFLD) using Swiss Prot Database perfectly matched with calreticulin (CRT), a major microsomal calcium binding protein of the endoplasmic reticulum (ER). The identity of TAase with CRT was substantiated by the observation that the purified TAase avidly reacted with commercially available antibody raised against the C-terminus of human CRT (13 residues peptide, DEEDATGQAKDEL). Purified TAase also showed Ca2+ binding and acted as a substrate for phosphorylation catalyzed by protein kinase C (PKC), which are hallmark characteristics of CRT. Further, purified placental CRT as well as the commercially procured pure CRT yielded significant TAase catalytic activity and were also found effective in mediating the acetylation of the target protein NADPH cytochrome P-450 reductase by DAMC as detected by Western blot using anti-acetyl lysine antibody. These observations for the first time convincingly attribute the transacetylase function to CRT. Hence, this transacetylase function of CRT is designated calreticulin transacetylase (CRTAase). We envisage that CRTAase plays an important role in protein modification by way of acetylation independent of Acetyl CoA.

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Authors and Affiliations

  1. Biochemistry Department, V.P. Chest Institute, University of Delhi, 110007, Delhi

    Seema, Ranju Kumari, Garima Gupta, Ajit Kumar, Sanjay Goel, Yogesh K. Tyagi, Ruchika Gulati, Anjali Vinocha & Hanumantharao G. Raj

  2. Department of Chemistry, University of Delhi, 110007, Delhi

    Ramesh C. Rastogi & Virinder S. Parmar

  3. Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, 110007, Delhi

    Daman Saluja

  4. Department of Zoology, University of Delhi, 110007, Delhi

    Kambadoor Muralidhar

  5. Institute of Nuclear Medicine and Allied Sciences, Lucknow Road, Timarpur, Delhi, India

    Bilikere S. Dwarakanth

  6. Department of Protein Biochemistry and Enzyme Design, Novozymes, A/S, Smormosvej 25, DK2880, Bagsvaerd, Denmark

    Shamkant A. Patkar

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  1. Seema
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Correspondence to Hanumantharao G. Raj.

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Seema, Kumari, R., Gupta, G. et al. Characterization of protein transacetylase from human placenta as a signaling molecule calreticulin using polyphenolic peracetates as the acetyl group donors. Cell Biochem Biophys 47, 53–64 (2007). https://doi.org/10.1385/CBB:47:1:53

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