Molecular and functional characterization of polymorphisms in the secreted phospholipase A2 group X gene: relevance to coronary artery disease

Abstract

Among secreted phospholipases A2 (sPLA2s), human group X sPLA2 (hGX sPLA2) is emerging as a novel attractive therapeutic target due to its implication in inflammatory diseases. To elucidate whether hGX sPLA2 plays a causative role in coronary artery disease (CAD), we screened the human PLA2G10 gene to identify polymorphisms and possible associations with CAD end-points in a prospective study, AtheroGene. We identified eight polymorphisms, among which, one non-synonymous polymorphism R38C in the propeptide region of the sPLA2. The T-512C polymorphism located in the 5' untranslated region was associated with a decreased risk of recurrent cardiovascular events during follow-up. The functional analysis of the R38C polymorphism showed that it leads to a profound change in expression and activity of hGX sPLA2, although there was no detectable impact on CAD risk. Due to the potential role of hGX sPLA2 in inflammatory processes, these polymorphisms should be investigated in other inflammatory diseases.

Fig. 1

Schematic representation of the human PLA2G10 gene and the position of SNPs. Exons are shown as vertical bars. Intron size is indicated. Exons 1–4 encode for the 123 amino acid hGX sPLA2 protein. The polymorphism that resulted in arginine to cysteine substitution is shown in bold

Fig. 2

a Schematic representation of the hGX sPLA2 protein containing the R38C polymorphism in the propeptide region. The arginine doublet preceding the mature protein sequence is indicated in bold. The arrow indicates the allelic substitution, and the amino acid change (arginine R to cysteine C) is indicated in red. b Immunofluorescence localization of hGX sPLA2 in COS-7 cells transfected with vectors encoding the R38 and the C38 proteins. The hGX sPLA2 protein is labeled with red. Nucleus is labeled in blue with DAPI. Confocal analysis was performed on a Leica SP2-AOBS confocal microscope as described in “Materials and methods”. Magnification: 50

Fig. 3

Subcellular immunofluorescence localization of hGX sPLA2 in COS-7 cells transiently transfected with vectors encoding either (a) R38 or (b) C38 hGX sPLA2. Twenty hours after transfection cells were fixed and stained with rabbit polyclonal against hGX sPLA2 (red) and either a mouse monoclonal against the ER marker PDI (green), or a mouse monoclonal against the Golgi apparatus marker 58 K protein (green). Nucleus is labelled in blue with DAPI Confocal analysis was performed on a Leica SP2-AOBS confocal microscope as described in “Materials and methods”. Magnification 50 for ER and 100 for Golgi

Fig. 4

a hGX sPLA2 enzymatic activity measured with radiolabeled Escherichia coli membranes in supernatant and cell lysates of COS-7 cells transfected either with an empty vector or with vectors encoding the R38 or C38 proteins as described in “Materials and methods”. A representative experiment of at least three experiments with similar results is shown. b Concentration of hGX sPLA2 protein quantified in supernatant and cell lysates of COS-7 cells transfected either with an empty vector or with vectors encoding the R38 or C38 proteins using TR-FIA assay as described in “Materials and methods”. A representative experiment of at least three experiments with similar results is shown

Fig. 5

Effect of temperature cell growth on the expression of hGX sPLA2. COS-7 cells transfected with either R38- or C38-containing expression vectors were grown at 30°C or 37°C for 72 h. The total amount of hGX sPLA2 protein was then quantified in the cell lysate (a) and supernatant (b) using the TR-FIA assay as described in “Materials and methods”. c COS-7 cells transfected with either the R38- or C38-containing or empty expression vectors were grown at 30°C or 37°C for 72 h, and hGX sPLA2 activity was measured in cell supernatants as described in “Materials and methods”. Cells transfected with the empty vector showed no detectable hGX sPLA2 protein or enzyme activity (not shown)