Calreticulin transacetylase (CRTAase): Identification of novel substrates and CRTAase-mediated modification of protein kinase C (PKC) activity in lymphocytes of asthmatic patients by polyphenolic acetates

Gulati, Ruchika; Kumar, Ajit; Bansal, Seema; Tyagi, Yogesh K.; Tyagi, Tapesh K.; Ponnan, Prija; Malhotra, Shashwat; Jain, Sapan K.; Singh, Usha; Bansal, Surendra K.; Raj, Hanumantharao G.; Dwarakanath, Bilikere S.; Chaudhury, Nabo K.; Vij, Anjana; Vijayan, Vannan K.; Rastogi, Ramesh C.; Parmar, Virinder S.

doi:10.1351/pac200779040729

Pure Appl. Chem., 2007, Vol. 79, No. 4, pp. 729-737

http://dx.doi.org/10.1351/pac200779040729

Calreticulin transacetylase (CRTAase): Identification of novel substrates and CRTAase-mediated modification of protein kinase C (PKC) activity in lymphocytes of asthmatic patients by polyphenolic acetates

Ruchika Gulati¹, Ajit Kumar¹, Seema Bansal¹, Yogesh K. Tyagi¹, Tapesh K. Tyagi¹, Prija Ponnan¹, Shashwat Malhotra², Sapan K. Jain², Usha Singh¹, Surendra K. Bansal¹, Hanumantharao G. Raj¹, Bilikere S. Dwarakanath³, Nabo K. Chaudhury³, Anjana Vij⁴, Vannan K. Vijayan¹, Ramesh C. Rastogi² and Virinder S. Parmar²

¹ Department of Biochemistry, V. P. Chest Institute, University of Delhi, Delhi-110 007, India
² Department of Chemistry, University of Delhi, Delhi-110 007, India
³ Institute of Nuclear Medicine and Allied Sciences, Lucknow Road, Delhi-110 007, India
⁴ Defence Institute of Physiology and Applied Sciences, DRDO, Lucknow Road, Delhi-110007, India

Abstract: Earlier reports from our laboratory established the acetyl transferase function of calreticulin (CRT), enabling CRT to transfer acetyl groups from the acetoxy groups of polyphenolic acetates (PAs) to certain receptor proteins. We have in this paper documented the ability of CRT to catalyze the possible transfer of acetyl moiety from 7-acetamido-4-methylcoumarin (7-N-AMC) to the proteins, glutathione S-transferase (GST), and NADPH cytochrome c reductase, leading to the modification of their catalytic activities. 7-Acetoxy-4-methylthiocoumarin (7-AMTC) compared to 7-acetoxy-4-methylcoumarin (7-AMC) when used as a substrate for calreticulin transacetylase (CRTAase) yielded significantly higher catalytic activity. PM3-optimized geometries suggested that the availability of electrons on the sulfur atom of the thiocarbonyl group of the thiocoumarin may render the substrate binding more favorable to the active site of the enzyme as compared to its oxygen analog. Further CRTAase activity was characterized in the human blood lymphocytes. There was no appreciable difference in CRTAase activity of lymphocytes of asthmatic patients as compared to those of normal subjects. The results presented here highlight for the first time the irreversible inhibition of human blood lymphocytes protein kinase C (PKC) by 7,8-diacetoxy-4-methylcoumarin (DAMC) possibly by way of acetylation. The activity of PKC in lymphocytes of asthmatic patients was found to proportionally increase with the severity of the disease. When PA was incubated with lymphocytes of normal patients, PKC was inhibited marginally. On the other hand, lymphocyte PKC of severe asthmatic patients was inhibited drastically. Several PAs inhibited PKC of asthmatic patients in tune with their specificity to CRTAase. DAMC was found to exert maximum inhibitory action on PKC, while 7,8-dihydroxy-4-methylcoumarin (DHMC), the deacetylated product of DAMC, failed to inhibit PKC. These observations clearly describe DAMC as the novel irreversible inhibitor of PKC, and DAMC may be found useful in the control of inflammation and may serve as a potential drug candidate in the therapy of asthma.