Effect of E54 mutation of human secreted phospholipase A2 GIIE on substrate selectivity.

Shulin Hou; Junping Bai; Xin LU; Yulong Zhang; Tingting XU; Jun Xie

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Effect of E54 mutation of human secreted phospholipase A2 GIIE on substrate selectivity.

Author: Shulin HOU ¹ ; Junping BAI ¹ ; Xin LU ¹ ; Yulong ZHANG ² ; Tingting XU ² ; Jun XIE ¹
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1. Shanxi Key Laboratory of Birth Defect and Cell Regeneration, College of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001, Shanxi, China.
2. State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, Guangdong, China.
Publication Type:Journal Article
Keywords: GIIE; mutation; recombinant expression; secreted phospholipase A2; substrate head-group selectivity
MeSH: Humans; Kinetics; Mutation; Phospholipases A2, Secretory; Phospholipids; Saccharomycetales; Substrate Specificity
From: Chinese Journal of Biotechnology 2021;37(7):2513-2521
CountryChina
Language:Chinese
Abstract: Human secreted phospholipase A2 GIIE (hGIIE) is involved in inflammation and lipid metabolism due to its ability of hydrolyzing phospholipids. To reveal the mechanism of substrate head-group selectivity, we analyzed the effect of mutation of hGIIE on its activity and selectivity. hGIIE structural analysis showed that E54 might be related to its substrate head-group selectivity. According to the sequence alignment, E54 was mutated to alanine, phenylalanine, and lysine. Mutated genes were cloned and expressed in Pichia pastoris X33, and the enzymes with mutations were purified with 90% purity by ion exchange and molecular size exclusion chromatography. The enzymatic activities were determined by isothermal microthermal titration method. The Km of mutant E54K towards 1,2-dihexyl phosphate glycerol decreased by 0.39-fold compared with that of wild type hGIIE (WT), and the Km of E54F towards 1,2-dihexanoyl-sn-glycero-3-phosphocholine increased by 1.93-fold than that of WT. The affinity of mutant proteins with phospholipid substrate was significantly changed, indicating that E54 plays an important role in the substrate head-group selectivity of hGIIE.