PAF-AH and PLA2 activity of Leptospira interrogans LA_2144 gene product

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 extracted 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 hydrolyze 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 superfamily, 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. Expression 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 detected. Conclusions L. interrogans LA_2144 gene product had a stronger PAF-AH and a certain PLA2 activity, which might involve in the hemorrhage and inflammatory response in leptospirosis. Key words: Leptospira interrogans; LA_2144 gene; PAF-AH; PLA2; Enzymatic activity