Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 Feb 15;306(Pt 1):167–175. doi: 10.1042/bj3060167

Phospholipase A2 from plasma of patients with septic shock is associated with high-density lipoproteins and C3 anaphylatoxin: some implications for its functional role.

M A Gijón 1, C Pérez 1, E Méndez 1, M Sánchez Crespo 1
PMCID: PMC1136497  PMID: 7864806

Abstract

Phospholipase A2 (PLA2) activity was purified 12,544-fold with a 13% yield from the plasma of patients diagnosed of septic shock by the sequential use of heparin-agarose affinity chromatography, gel filtration, and reverse-phase f.p.l.c. Gel-filtration chromatography of plasma omitting high-ionic-strength buffer revealed a molecular mass different from that of purified PLA2 and co-elution with apolipoprotein A-I peaks, which suggests its association with high-density lipoproteins (HDL). N-terminal analysis of the enzyme activity protein band, electroblotted from a SDS-acrylamide gel and with an assessed molecular mass of 19 kDa, showed an identical sequence to that of alpha-chain of human C3 complement component, suggesting the presence in this band of a complex formed by a complement C3-derived anaphylatoxin (C3a)-related fragment and the PLA2 linked side-by-side. Because the preparation of plasma enzyme showed lower activity than the enzyme obtained from fibroblasts transfected with the coding sequence of human group-II PLA2, and because the addition of C3-derived anaphylatoxins from human serum inhibited the activity of this recombinant PLA2, it was considered that C3a-related peptides behave as inhibitors of group-II PLA2. The enzyme showed optimal activity on [14C]oleate-labelled autoclaved E. coli, on synthetic phosphatidylethanolamine, and on [3H]arachidonate-labelled membranes of the monoblast cell line U937, but it did not show any activity on the release of [3H]arachidonate from pre-labelled human polymorphonuclear leukocytes (PMNs). In short, PLA2 from plasma of sepsis patients shows unique associations with other plasma proteins which may influence its functional properties. The association with C3-related peptides shows an inhibitory effect on the enzyme activity, whereas the association with HDL might influence its environment and/or its interaction with cells. The study of the catalytic properties shows a prominent effect on bacterial phospholipids, synthetic phosphatidylethanolamine, and membranes from U937 monoblasts, but not on synthetic phosphatidylcholine or on PMNs, even when these cells were maintained in culture to allow spontaneous apoptosis and became a good substrate for pancreatic type PLA2.

Full text

PDF
167

Images in this article

171Figure 3
on p.171

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aarsman A. J., Leunissen-Bijvelt J., Van den Koedijk C. D., Neys F. W., Verkleij A. J., Van den Bosch H. Phospholipase A2 activity in platelets. Immuno-purification and localization of the enzyme in rat platelets. J Lipid Mediat. 1989 Jan-Feb;1(1):49–61. [PubMed] [Google Scholar]
  2. Alper C. A., Johnson A. M., Birtch A. G., Moore F. D. Human C'3: evidence for the liver as the primary site of synthesis. Science. 1969 Jan 17;163(3864):286–288. doi: 10.1126/science.163.3864.286. [DOI] [PubMed] [Google Scholar]
  3. Asaoka Y., Yoshida K., Sasaki Y., Nishizuka Y., Murakami M., Kudo I., Inoue K. Possible role of mammalian secretory group II phospholipase A2 in T-lymphocyte activation: implication in propagation of inflammatory reaction. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):716–719. doi: 10.1073/pnas.90.2.716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  5. Barbour S. E., Dennis E. A. Antisense inhibition of group II phospholipase A2 expression blocks the production of prostaglandin E2 by P388D1 cells. J Biol Chem. 1993 Oct 15;268(29):21875–21882. [PubMed] [Google Scholar]
  6. Bone R. C., Fisher C. J., Jr, Clemmer T. P., Slotman G. J., Metz C. A., Balk R. A. Sepsis syndrome: a valid clinical entity. Methylprednisolone Severe Sepsis Study Group. Crit Care Med. 1989 May;17(5):389–393. [PubMed] [Google Scholar]
  7. Boni L. T., Hui S. W. Polymorphic phase behaviour of dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine mixtures. Structural changes between hexagonal, cubic and bilayer phases. Biochim Biophys Acta. 1983 Jun 10;731(2):177–185. doi: 10.1016/0005-2736(83)90007-x. [DOI] [PubMed] [Google Scholar]
  8. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  9. Chen C. H., Forte T. H., Cahoon B. E., Thrift R. N., Albers J. J. Synthesis and secretion of lecithin-cholesterol acyltransferase by the human hepatoma cell line HepG2. Biochim Biophys Acta. 1986 Jul 18;877(3):433–439. doi: 10.1016/0005-2760(86)90209-2. [DOI] [PubMed] [Google Scholar]
  10. Chen J., Engle S. J., Seilhamer J. J., Tischfield J. A. Cloning and recombinant expression of a novel human low molecular weight Ca(2+)-dependent phospholipase A2. J Biol Chem. 1994 Jan 28;269(4):2365–2368. [PubMed] [Google Scholar]
  11. Clark J. D., Lin L. L., Kriz R. W., Ramesha C. S., Sultzman L. A., Lin A. Y., Milona N., Knopf J. L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell. 1991 Jun 14;65(6):1043–1051. doi: 10.1016/0092-8674(91)90556-e. [DOI] [PubMed] [Google Scholar]
  12. Connor J., Bucana C., Fidler I. J., Schroit A. J. Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation. Proc Natl Acad Sci U S A. 1989 May;86(9):3184–3188. doi: 10.1073/pnas.86.9.3184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Crowl R. M., Stoller T. J., Conroy R. R., Stoner C. R. Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response. J Biol Chem. 1991 Feb 5;266(4):2647–2651. [PubMed] [Google Scholar]
  14. Davidson F. F., Dennis E. A., Powell M., Glenney J. R., Jr Inhibition of phospholipase A2 by "lipocortins" and calpactins. An effect of binding to substrate phospholipids. J Biol Chem. 1987 Feb 5;262(4):1698–1705. [PubMed] [Google Scholar]
  15. Dennis E. A., Rhee S. G., Billah M. M., Hannun Y. A. Role of phospholipase in generating lipid second messengers in signal transduction. FASEB J. 1991 Apr;5(7):2068–2077. doi: 10.1096/fasebj.5.7.1901288. [DOI] [PubMed] [Google Scholar]
  16. Elsbach P., Weiss J., Franson R. C., Beckerdite-Quagliata S., Schneider A., Harris L. Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship. J Biol Chem. 1979 Nov 10;254(21):11000–11009. [PubMed] [Google Scholar]
  17. Fonteh A. N., Chilton F. H. Rapid remodeling of arachidonate from phosphatidylcholine to phosphatidylethanolamine pools during mast cell activation. J Immunol. 1992 Mar 15;148(6):1784–1791. [PubMed] [Google Scholar]
  18. Gassama-Diagne A., Fauvel J., Chap H. Purification of a new, calcium-independent, high molecular weight phospholipase A2/lysophospholipase (phospholipase B) from guinea pig intestinal brush-border membrane. J Biol Chem. 1989 Jun 5;264(16):9470–9475. [PubMed] [Google Scholar]
  19. Green J. A., Smith G. M., Buchta R., Lee R., Ho K. Y., Rajkovic I. A., Scott K. F. Circulating phospholipase A2 activity associated with sepsis and septic shock is indistinguishable from that associated with rheumatoid arthritis. Inflammation. 1991 Oct;15(5):355–367. doi: 10.1007/BF00917352. [DOI] [PubMed] [Google Scholar]
  20. Gronich J. H., Bonventre J. V., Nemenoff R. A. Purification of a high-molecular-mass form of phospholipase A2 from rat kidney activated at physiological calcium concentrations. Biochem J. 1990 Oct 1;271(1):37–43. doi: 10.1042/bj2710037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Habenicht A. J., Salbach P., Goerig M., Zeh W., Janssen-Timmen U., Blattner C., King W. C., Glomset J. A. The LDL receptor pathway delivers arachidonic acid for eicosanoid formation in cells stimulated by platelet-derived growth factor. Nature. 1990 Jun 14;345(6276):634–636. doi: 10.1038/345634a0. [DOI] [PubMed] [Google Scholar]
  22. Hanasaki K., Arita H. Characterization of a high affinity binding site for pancreatic-type phospholipase A2 in the rat. Its cellular and tissue distribution. J Biol Chem. 1992 Mar 25;267(9):6414–6420. [PubMed] [Google Scholar]
  23. Hugli T. E., Vallota E. H., Müller-Eberhard H. J. Purification and partial characterization of human and porcine C3a anaphylatoxin. J Biol Chem. 1975 Feb 25;250(4):1472–1478. [PubMed] [Google Scholar]
  24. Inada M., Tojo H., Kawata S., Tarui S., Okamoto M. Induction of group II-like phospholipase A2 by lipopolysaccharide in the liver of BCG-primed rat. Biochem Biophys Res Commun. 1991 Feb 14;174(3):1077–1083. doi: 10.1016/0006-291x(91)91530-p. [DOI] [PubMed] [Google Scholar]
  25. Jain M. K., Yu B. Z., Kozubek A. Binding of phospholipase A2 to zwitterionic bilayers is promoted by lateral segregation of anionic amphiphiles. Biochim Biophys Acta. 1989 Mar 27;980(1):23–32. doi: 10.1016/0005-2736(89)90195-8. [DOI] [PubMed] [Google Scholar]
  26. Jiao S., Cole T. G., Kitchens R. T., Pfleger B., Schonfeld G. Genetic heterogeneity of lipoproteins in inbred strains of mice: analysis by gel-permeation chromatography. Metabolism. 1990 Feb;39(2):155–160. doi: 10.1016/0026-0495(90)90069-o. [DOI] [PubMed] [Google Scholar]
  27. Kanda A., Ono T., Yoshida N., Tojo H., Okamoto M. The primary structure of a membrane-associated phospholipase A2 from human spleen. Biochem Biophys Res Commun. 1989 Aug 30;163(1):42–48. doi: 10.1016/0006-291x(89)92096-2. [DOI] [PubMed] [Google Scholar]
  28. Kim D. K., Kudo I., Inoue K. Purification and characterization of rabbit platelet cytosolic phospholipase A2. Biochim Biophys Acta. 1991 Apr 24;1083(1):80–88. doi: 10.1016/0005-2760(91)90127-4. [DOI] [PubMed] [Google Scholar]
  29. Knaus W. A., Draper E. A., Wagner D. P., Zimmerman J. E. APACHE II: a severity of disease classification system. Crit Care Med. 1985 Oct;13(10):818–829. [PubMed] [Google Scholar]
  30. Kramer R. M., Pepinsky R. B. Assay and purification of phospholipase A2 from human synovial fluid in rheumatoid arthritis. Methods Enzymol. 1991;197:373–381. doi: 10.1016/0076-6879(91)97163-s. [DOI] [PubMed] [Google Scholar]
  31. Kramer R. M., Roberts E. F., Manetta J., Putnam J. E. The Ca2(+)-sensitive cytosolic phospholipase A2 is a 100-kDa protein in human monoblast U937 cells. J Biol Chem. 1991 Mar 15;266(8):5268–5272. [PubMed] [Google Scholar]
  32. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  33. Lai C. Y., Wada K. Phospholipase A2 from human synovial fluid: purification and structural homology to the placental enzyme. Biochem Biophys Res Commun. 1988 Dec 15;157(2):488–493. doi: 10.1016/s0006-291x(88)80275-4. [DOI] [PubMed] [Google Scholar]
  34. Leslie C. C., Voelker D. R., Channon J. Y., Wall M. M., Zelarney P. T. Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7. Biochim Biophys Acta. 1988 Dec 16;963(3):476–492. doi: 10.1016/0005-2760(88)90316-5. [DOI] [PubMed] [Google Scholar]
  35. Mayer R. J., Marshall L. A. New insights on mammalian phospholipase A2(s); comparison of arachidonoyl-selective and -nonselective enzymes. FASEB J. 1993 Feb 1;7(2):339–348. doi: 10.1096/fasebj.7.2.8440410. [DOI] [PubMed] [Google Scholar]
  36. Morgan D. W., Anderson C. M., Meyers K. P. Recombinant human type II phospholipase A2 lacks edema producing activity in rat. Eur J Pharmacol. 1993 Apr 22;235(1):45–50. doi: 10.1016/0014-2999(93)90818-3. [DOI] [PubMed] [Google Scholar]
  37. Morris K. M., Aden D. P., Knowles B. B., Colten H. R. Complement biosynthesis by the human hepatoma-derived cell line HepG2. J Clin Invest. 1982 Oct;70(4):906–913. doi: 10.1172/JCI110687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Murakami M., Hara N., Kudo I., Inoue K. Triggering of degranulation in mast cells by exogenous type II phospholipase A2. J Immunol. 1993 Nov 15;151(10):5675–5684. [PubMed] [Google Scholar]
  39. Murakami M., Kudo I., Inoue K. Molecular nature of phospholipases A2 involved in prostaglandin I2 synthesis in human umbilical vein endothelial cells. Possible participation of cytosolic and extracellular type II phospholipases A2. J Biol Chem. 1993 Jan 15;268(2):839–844. [PubMed] [Google Scholar]
  40. Murakami M., Kudo I., Nakamura H., Yokoyama Y., Mori H., Inoue K. Exacerbation of rat adjuvant arthritis by intradermal injection of purified mammalian 14-kDa group II phospholipase A2. FEBS Lett. 1990 Jul 30;268(1):113–116. doi: 10.1016/0014-5793(90)80986-s. [DOI] [PubMed] [Google Scholar]
  41. Op den Kamp J. A. Lipid asymmetry in membranes. Annu Rev Biochem. 1979;48:47–71. doi: 10.1146/annurev.bi.48.070179.000403. [DOI] [PubMed] [Google Scholar]
  42. Parthasarathy S., Barnett J. Phospholipase A2 activity of low density lipoprotein: evidence for an intrinsic phospholipase A2 activity of apoprotein B-100. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9741–9745. doi: 10.1073/pnas.87.24.9741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Perlmutter D. H., Dinarello C. A., Punsal P. I., Colten H. R. Cachectin/tumor necrosis factor regulates hepatic acute-phase gene expression. J Clin Invest. 1986 Nov;78(5):1349–1354. doi: 10.1172/JCI112721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Perlmutter D. H., Goldberger G., Dinarello C. A., Mizel S. B., Colten H. R. Regulation of class III major histocompatibility complex gene products by interleukin-1. Science. 1986 May 16;232(4752):850–852. doi: 10.1126/science.3010455. [DOI] [PubMed] [Google Scholar]
  45. Pernas P., Masliah J., Olivier J. L., Salvat C., Rybkine T., Bereziat G. Type II phospholipase A2 recombinant overexpression enhances stimulated arachidonic acid release. Biochem Biophys Res Commun. 1991 Aug 15;178(3):1298–1305. doi: 10.1016/0006-291x(91)91035-b. [DOI] [PubMed] [Google Scholar]
  46. Pfeilschifter J., Schalkwijk C., Briner V. A., van den Bosch H. Cytokine-stimulated secretion of group II phospholipase A2 by rat mesangial cells. Its contribution to arachidonic acid release and prostaglandin synthesis by cultured rat glomerular cells. J Clin Invest. 1993 Nov;92(5):2516–2523. doi: 10.1172/JCI116860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Qiu Z. H., de Carvalho M. S., Leslie C. C. Regulation of phospholipase A2 activation by phosphorylation in mouse peritoneal macrophages. J Biol Chem. 1993 Nov 15;268(32):24506–24513. [PubMed] [Google Scholar]
  48. Ramesha C. S., Ives D. L. Detection of arachidonoyl-selective phospholipase A2 in human neutrophil cytosol. Biochim Biophys Acta. 1993 May 20;1168(1):37–44. doi: 10.1016/0005-2760(93)90263-9. [DOI] [PubMed] [Google Scholar]
  49. Satoh K., Imaizumi T., Kawamura Y., Yoshida H., Hiramoto M., Takamatsu S., Takamatsu M. Platelet-activating factor (PAF) stimulates the production of PAF acetylhydrolase by the human hepatoma cell line, HepG2. J Clin Invest. 1991 Feb;87(2):476–481. doi: 10.1172/JCI115020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Savill J. S., Wyllie A. H., Henson J. E., Walport M. J., Henson P. M., Haslett C. Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest. 1989 Mar;83(3):865–875. doi: 10.1172/JCI113970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schalkwijk C. G., Märki F., Wiesenberg I., van den Bosch H. The detection of multimeric forms of phospholipase A2 upon sodium dodecylsulfate-polyacrylamide electrophoresis. J Lipid Mediat. 1991 Jul-Aug;4(1):83–95. [PubMed] [Google Scholar]
  52. Seilhamer J. J., Pruzanski W., Vadas P., Plant S., Miller J. A., Kloss J., Johnson L. K. Cloning and recombinant expression of phospholipase A2 present in rheumatoid arthritic synovial fluid. J Biol Chem. 1989 Apr 5;264(10):5335–5338. [PubMed] [Google Scholar]
  53. Stafforini D. M., Elstad M. R., McIntyre T. M., Zimmerman G. A., Prescott S. M. Human macrophages secret platelet-activating factor acetylhydrolase. J Biol Chem. 1990 Jun 15;265(17):9682–9687. [PubMed] [Google Scholar]
  54. Suwa Y., Kudo I., Imaizumi A., Okada M., Kamimura T., Suzuki Y., Chang H. W., Hara S., Inoue K. Proteinaceous inhibitors of phospholipase A2 purified from inflammatory sites in rats. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2395–2399. doi: 10.1073/pnas.87.7.2395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Symer D. E., Paznekas W. A., Shin H. S. A requirement for membrane-associated phospholipase A2 in platelet cytotoxicity activated by receptors for immunoglobulin G and complement. J Exp Med. 1993 Apr 1;177(4):937–947. doi: 10.1084/jem.177.4.937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Takayama K., Kudo I., Kim D. K., Nagata K., Nozawa Y., Inoue K. Purification and characterization of human platelet phospholipase A2 which preferentially hydrolyzes an arachidonoyl residue. FEBS Lett. 1991 May 6;282(2):326–330. doi: 10.1016/0014-5793(91)80506-x. [DOI] [PubMed] [Google Scholar]
  57. Vadas P. Elevated plasma phospholipase A2 levels: correlation with the hemodynamic and pulmonary changes in gram-negative septic shock. J Lab Clin Med. 1984 Dec;104(6):873–881. [PubMed] [Google Scholar]
  58. Vadas P., Stefanski E., Pruzanski W. Characterization of extracellular phospholipase A2 in rheumatoid synovial fluid. Life Sci. 1985 Feb 11;36(6):579–587. doi: 10.1016/0024-3205(85)90640-x. [DOI] [PubMed] [Google Scholar]
  59. Vadas P., Wasi S., Movat H. Z., Hay J. B. Extracellular phospholipase A2 mediates inflammatory hyperaemia. Nature. 1981 Oct 15;293(5833):583–585. doi: 10.1038/293583a0. [DOI] [PubMed] [Google Scholar]
  60. Wright G. C., Weiss J., Kim K. S., Verheij H., Elsbach P. Bacterial phospholipid hydrolysis enhances the destruction of Escherichia coli ingested by rabbit neutrophils. Role of cellular and extracellular phospholipases. J Clin Invest. 1990 Jun;85(6):1925–1935. doi: 10.1172/JCI114655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wright G. W., Ooi C. E., Weiss J., Elsbach P. Purification of a cellular (granulocyte) and an extracellular (serum) phospholipase A2 that participate in the destruction of Escherichia coli in a rabbit inflammatory exudate. J Biol Chem. 1990 Apr 25;265(12):6675–6681. [PubMed] [Google Scholar]
  62. de Bruijn M. H., Fey G. H. Human complement component C3: cDNA coding sequence and derived primary structure. Proc Natl Acad Sci U S A. 1985 Feb;82(3):708–712. doi: 10.1073/pnas.82.3.708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. van den Bosch H. Intracellular phospholipases A. Biochim Biophys Acta. 1980 Sep 30;604(2):191–246. doi: 10.1016/0005-2736(80)90574-x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES