Joshi Rini, Singh Prabhjot, Sharma Naresh K, Ponnan Prija, Saluja Daman, Gambhir Jasvinder K, Rawat Diwan S, Parmar Virinder S, Dwarakanath Bilkere S, Prasad Ashok K, Raj Hanumantharao G
Department of Biochemistry, V.P. Chest Institute, Delhi, India.
Department of Chemistry, University of Delhi, Delhi, 110 007 India.
3 Biotech. 2021 Mar;11(3):113. doi: 10.1007/s13205-021-02659-1. Epub 2021 Feb 3.
In silico-docking studies from previous work have suggested that Lys-206 and lys-207 of calreticulin (CR) play a pivotal key role in its well-established transacetylation activity. To experimentally validate this prediction, we introduced three mutations at lysine residues of P-domain of CR: K → A, (K -206, -209), (K -206, -207) and (K -207, -209) and analyzed their immunoreactivity and acetylation potential. The clones of wild-type P-domain ( ) and three mutated P-domain ( , and ) were expressed in pTrcHis C vector and the recombinant , , and proteins were purified by Ni-NTA affinity chromatography. Screening of the transacylase activity (TAase) by the Glutathione S Transferase (GST) assay revealed that the TAase activity was associated with the and while and did not show any activity. The immune-reactivity to anti-lysine antibody was also retained only by the in which the Lys-207 was intact. Retention of the TAase activity and immunoreactivity of with mutations introduced at Lys-206, Lys-209, while its loss with a mutation at Lys-207 residue indicated that lysine-207 of P-domain constitutes the active site residue controlling TAase activity.
The online version contains supplementary material available at 10.1007/s13205-021-02659-1.
先前工作中的计算机对接研究表明，钙网蛋白（CR）的赖氨酸-206和赖氨酸-207在其已确立的转乙酰化活性中起关键作用。为了通过实验验证这一预测，我们在CR的P结构域的赖氨酸残基处引入了三个突变：K→A，（K -206，-209），（K -206，-207）和（K -207，-209），并分析了它们的免疫反应性和乙酰化潜力。野生型P结构域（ ）和三个突变的P结构域（ 、 和 ）的克隆在pTrcHis C载体中表达，重组的 、 、 和 蛋白通过镍-氮三乙酸亲和层析法纯化。通过谷胱甘肽S-转移酶（GST）测定筛选转酰基酶活性（TAase），结果显示TAase活性与 和 相关，而 和 未显示任何活性。仅赖氨酸-207完整的 保留了对抗赖氨酸抗体的免疫反应性。在赖氨酸-206、赖氨酸-209处引入突变的 保留了TAase活性和免疫反应性，而在赖氨酸-207残基处引入突变则使其丧失，这表明P结构域的赖氨酸-207构成了控制TAase活性的活性位点残基。
在线版本包含可在10.1007/s13205-021-02659-1获取的补充材料。
