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.
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.
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DOI：
10.2174/092986611794927938
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发表时间：
2011-05
影响因子：
1.6
通讯作者：
Prabhjot Singh;Prija Ponnan;N. Priya;T. K. Tyagi;M. Gaspari;Shibu Krishnan;G. Cuda;P. Joshi;J. Gambhir;Sunil K. Sharma;A. Prasad;L. Saso;R. C. Rastogi;V. Parmar;H. Raj
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作者：
Prabhjot Singh;Prija Ponnan;N. Priya;T. K. Tyagi;M. Gaspari;Shibu Krishnan;G. Cuda;P. Joshi;J. Gambhir;Sunil K. Sharma;A. Prasad;L. Saso;R. C. Rastogi;V. Parmar;H. Raj
钙网蛋白转酰酶（calreticulin transacylase，CRTAase）是内质网（endoplasmic reticulum，ER）蛋白中催化酰基从酰氧基香豆素转移到受体蛋白的一种独特的生化功能。本研究，不像以前的报告，其他人利用CR缺陷细胞单独，处理的重组CR域的捻转血矛线虫（rhCRTAase），以检查其CRTAase活性。与N-和C-结构域不同，rhCR的P-结构域被发现具有CRTAase功能。我们还首次观察到乙酰CoA作为rhCRTAase/P结构域介导的重组日本血吸虫谷胱甘肽S-转移酶（rGST）乙酰化的底物。还发现rhCRTAase/P-结构域通过酰氧基香豆素进行自酰化。此外，单独的非变性形式的分离的自酰化rhCRTAase/P-结构域表现出将酰基转移至rGST的能力，表明酰化CR的稳定中间体性质。通过LC-MS/MS分析证明，P-结构域催化的rGST被7，8-二乙酰氧基-4-甲基香豆素或乙酰CoA乙酰化，导致几个共同的赖氨酸残基的修饰。通过计算盲对接研究预测了酰氧基香豆素与CR结合的推定位点。结果显示，P-结构域中存在的两个赖氨酸残基Lys-173和Lys-174参与结合酰氧基香豆素和乙酰CoA，因此突出了CRTA酶活性的活性位点将位于CR的P-结构域中。已知某些ER蛋白在涉及乙酰辅酶A的生理条件下经历乙酰化。这些结果表明，CRTAase介导的乙酰CoA蛋白乙酰化可能暗示CR可能是ER腔的蛋白乙酰转移酶。
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.