ABSTRACT
The endothelial protein C receptor (EPCR) plays an important role in cardiovascular disease by binding protein C/activated protein C (APC). EPCR structure contains a hydrophobic groove filled with an unknown phospholipid needed to perform its function. It has not been established whether lipid exchange takes place in EPCR as a regulatory mechanism of its activity. Our objective was to identify this phospholipid and to explore the possibility of lipid exchange as a regulatory mechanism of EPCR activity driven by the endothelially expressed secretory group V phospholipase A(2) (sPLA(2)-V). We identified phosphatidylcholine (PCh) as the major phospholipid bound to human soluble EPCR (sEPCR). PCh in EPCR could be exchanged for lysophosphatidylcholine (lysoPCh) and platelet activating factor (PAF). Remarkably, lysoPCh and PAF impaired the protein C binding ability of sEPCR. Inhibition of sPLA(2)-V, responsible for lysoPCh and PAF generation, improved APC binding to endothelial cells. EPCR-dependent protein C activation and APC antiapoptotic effect were thus significantly enhanced. In contrast, endothelial cell supplementation with sPLA(2)-V inhibited both APC generation and its antiapoptotic effects. We conclude that APC generation and function can be modulated by changes in phospholipid occupancy of its endothelial cell receptor.
Subject(s)
Antigens, CD/chemistry , Antigens, CD/metabolism , Group V Phospholipases A2/metabolism , Lysophosphatidylcholines/chemistry , Platelet Activating Factor/chemistry , Protein C/metabolism , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Animals , Chromatography, Thin Layer , Endothelial Cells/metabolism , Endothelial Protein C Receptor , Enzyme Activation/physiology , Flow Cytometry , Humans , Immunohistochemistry , Lysophosphatidylcholines/metabolism , Mass Spectrometry , Mice , Platelet Activating Factor/metabolism , Protein Structure, Tertiary , Spectrometry, FluorescenceABSTRACT
Some physicochemical and rheological properties of the exopolysaccharide (EPS) produced by Pediococcus parvulus 2.6 were examined. Structural characterization by NMR ((1)H and 2D-COSY) showed that the same EPS, a 2-substituted (1,3)-beta-D-glucan, was synthesized irrespective of sugar source used for growth (glucose, fructose, or maltose). The molecular masses of these beta-glucans were always very high (>10(6) Da) and influenced by the culture medium or sugar source. The steady shear rheological experiments showed that all concentrations of the beta-glucan aqueous solutions exhibited a pseudoplastic behavior at high shear rates. Viscoelastic behavior of beta-glucan solutions was determined by dynamic oscillatory analysis. A critical concentration of 0.35% associated with the appearance of entanglements was calculated. The beta-glucan adopts an ordered hydrogen bond dependent helical conformation in neutral and slightly alkaline aqueous solutions, which was partly denatured under more alkaline conditions.