PAF-AH and PLA2 activity of Leptospira interrogans LA_2144 gene product
10.3760/cma.j.issn.0254-5101.2019.07.006
- VernacularTitle:问号钩端螺旋体LA_2144基因产物PAF-AH和PLA2酶活性研究
- Author:
Qinchao ZHANG
1
;
Aihua SUN
;
Jinliang ZHANG
;
Weilin HU
;
Jie YAN
;
Xu'ai LIN
Author Information
1. 浙江大学医学院病原生物学和微生物学系
- Keywords:
Leptospira interrogans;
LA_2144 gene;
PAF-AH;
PLA2;
Enzymatic activity
- From:
Chinese Journal of Microbiology and Immunology
2019;39(7):519-526
- CountryChina
- Language:Chinese
-
Abstract:
Objective To analyze the enzymatic activity of Leptospira interrogans ( L. interrogans) LA_2144 gene product to hydrolyze platelet activating factor acetylhydrolase ( PAF-AH) and phosphatidase A2(PLA2). Methods Bioinformatic softwares were used to predict transmembrane regions, signal peptides and domains of the LA_2144 gene of L. interrogans strain Lai. A prokaryotic expression system for signal peptide-free LA_2144 gene was established. The expressed target recombinant protein rLep2144 was extrac-ted by Ni-NTA affinity chromatography and then renatured. Spectrometry was used to detect the activity of rLep2144 to hydrolyze PAF-AH substrate 2-thio PAF and the Km and Kcat values as well as the activity to hy-drolyze PLA2 substrate arachidonoyl 2-thio PC. Real-time fluorescence quantitative RT-PCR and Western blot were performed to detect the transcription, protein expression and secretion of LA_2144 gene during infection of human and mouse vascular endothelial cells ( HUVEC and EOMA) with L. interrogans. Results L. interrogans LA_2144 gene contained a signal peptide and a domain belonging to SGNH hydrolase super-family, but no transmembrane regions. The established prokaryotic expression system for signal peptide-free LA_2144 gene could efficiently express rLep2144. The extracted rLep2144 was shown as a single protein fragment in separation gel and then successfully renatured. rLep2144 had a stronger PAF-AH activity with the Km and Kcat values of 688. 235 μmol/L and 0. 976/s, but its PLA2 activity was relatively weak. Expres-sion of the LA_2144 gene at mRNA and protein levels in HUVEC and EOMA was rapidly increased after the cells were infected with L. interrogans (P<0. 05) and the secretion of LA_2144 gene product could be detec-ted. Conclusions L. interrogans LA_2144 gene product had a stronger PAF-AH and a certain PLA2 activi-ty, which might involve in the hemorrhage and inflammatory response in leptospirosis.
