Phospholipase A1 Member A Activates Fibroblast-like Synoviocytes through the Autotaxin-Lysophosphatidic Acid Receptor Axis

Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.

Involvement of the γ Isoform of cPLA2 in the Biosynthesis of Bioactive N-Acylethanolamines

Arachidonylethanolamide (anandamide) acts as an endogenous ligand of cannabinoid receptors, while other N-acylethanolamines (NAEs), such as palmitylethanolamide and oleylethanolamide, show analgesic, anti-inflammatory, and appetite-suppressing effects through other receptors. In mammalian tissues, NAEs, including anandamide, are produced from glycerophospholipid via N-acyl-phosphatidylethanolamine (NAPE). The ɛ isoform of cytosolic phospholipase A2 (cPLA2) functions as an N-acyltransferase to form NAPE. Since the cPLA2 family consists of six isoforms (α, β, γ, δ, ɛ, and ζ), the present study investigated a possible involvement of isoforms other than ɛ in the NAE biosynthesis. Firstly, when the cells overexpressing one of the cPLA2 isoforms were labeled with [14C]ethanolamine, the increase in the production of [14C]NAPE was observed only with the ɛ-expressing cells. Secondly, when the cells co-expressing ɛ and one of the other isoforms were analyzed, the increase in [14C]N-acyl-lysophosphatidylethanolamine (lysoNAPE) and [14C]NAE was seen with the combination of ɛ and γ isoforms. Furthermore, the purified cPLA2γ hydrolyzed not only NAPE to lysoNAPE, but also lysoNAPE to glycerophospho-N-acylethanolamine (GP-NAE). Thus, the produced GP-NAE was further hydrolyzed to NAE by glycerophosphodiesterase 1. These results suggested that cPLA2γ is involved in the biosynthesis of NAE by its phospholipase A1/A2 and lysophospholipase activities.

Immobilization of Phospholipase A1 Using a Protein-Inorganic Hybrid System

In this study, four kinds of phospholipase A1-metal (Al/Co/Cu/Mn) hybrid nanostructures were prepared for enhancing the stability of the free PLA1. The formed hybrid complexes were characterized by scanning electron microscope (SEM), Fourier infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The stability and substrate specificity of immobilized enzymes were subsequently determined. After immobilization, the temperature tolerance of PLA1–metal hybrid nanostructures was enhanced. The relative activity of PLA1–Al/Co/Cu hybrid nanostructures remained above 60% at 50 °C, while that of free enzyme was below 5%. The thermal transition temperature measured by differential scanning calorimetry (DSC) was found to increase from 65.59 °C (free enzyme) to 173.14 °C, 123.67 °C, 96.31 °C, and 114.79 °C, referring to PLA1–Cu/Co/Al/Mn hybrid nanostructures, respectively. Additionally, after a storage for fourteen days at 4 °C, the immobilized enzymes could exhibit approximately 60% of the initial activity, while the free PLA1 was inactivated after four days of storage. In brief, using Co2+, Cu2+, Al3+, and Mn2+ as the hybridization materials for immobilization could improve the catalytic properties and stability of the free PLA1, suggesting a promising method for a wider application of PLA1 in many fields such as food, cosmetics, and the pharmaceutical industry.

Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.

GPR34 activation potentially bridges lymphoepithelial lesion to genesis of salivary gland MALT lymphoma

GPR34 translocation and mutation are specifically associated with salivary gland MALT lymphoma (SG-MALT-Lymphoma). Majority of GPR34 mutations are clustered in its C-terminus, resulting in truncated proteins lacking the phosphorylation motif important for receptor desensitization. It is unclear why GPR34 genetic changes associate with SG-MALT-Lymphoma and how these mutations contribute to the lymphoma development. We generated isogenic Flp-InTRex293 cell lines that stably expressed a single copy of GPR34 or its various mutants, and performed a range of in vitro assays. We showed that the GPR34 Q340X truncation, but not R84H and D151A mutants conferred a significantly increased resistance to apoptosis, and greater transforming potential than the GPR34 wild type. The GPR34 truncation mutant had a significantly delayed internalization than the wild type following ligand (lysophosphatidylserine) stimulation. Among 9 signaling pathways examined, the GPR34 Q340X truncation, to a lesser extent the D151A mutant, significantly activated CRE, NFkB and AP1 reporter activities, particularly in the presence of ligand stimulation. We further demonstrated enhanced activities of phospholipase-A1/2 in the culture supernatant of Flp-InTRex293 cells that expressed the GPR34 Q340X mutant, and their potential to catalyze the synthesis of lysophosphatidylserine from phosphatidylserine. Importantly, phospholipase-A1 was abundantly expressed in the duct epithelium of salivary glands and those involved in lymphoepithelial lesions (LELs). Our findings advocate a model of paracrine stimulation of malignant B-cells via GPR34, in which PLA is released by LELs, and hydrolyzes the phosphatidylserine exposed on apoptotic cells, generating lysophosphatidylserine, the ligand for GPR34. Thus, GPR34 activation potentially bridges LELs to genesis of SG-MALT-Lymphoma.