Structural basis for variations in the sensitivity of human decay accelerating factor to phosphatidylinositol-specific phospholipase C cleavage

E. I. Walter, W. L. Roberts, T. L. Rosenberry, W. D. Ratnoff, M. E. Medof

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105 Scopus citations

Abstract

Human decay-accelerating factor (DAF) proteins expressed on E and nucleated cells differ in their susceptibility to phosphatidylinositol (PI)-specific phospholipase C (PLC) cleavage/release. To investigate the mechanism of this difference, the glycoinositol-phospholipid anchoring moieties of E DAF, and of HeLa cell, polymorphonuclear cell, and lymphocyte DAF were structurally compared. Labeling of PI-PLC-resistant E DAF with 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)-diazirine ([125I]TID) and TLC analysis of nitrous acid deamination anchor fragments showed a predominant phospholipid species with less polar migration than the 125I-TID-labeled PI. Gas chromatographic analyses of methanolyzed E protein revealed 2.20 ± 0.16 mol of fatty acids [16:0, 18:0, 18:1, 20:4, 22:4, and 22:5 (0.76, 0.36, 0.25, 0.15, 0.40, 0.28 mol, respectively)] and 0.86 ± 0.05 mol of inositol per mol of N-terminal Asp. Gas chromatography-mass spectroscopy demonstrated principally myo-inositol but also variable amounts of the chiro-isomer. Nondenaturing polyacrylamide gel electrophoresis of 14C-radiomethylated E protein revealed that pretreatment with hydroxylamine, a reagent which removes ester-linked lipids, rendered it PI-PLC susceptible. In contrast, parallel analyses of 35S-cys-labeled PI-PLC-sensitive HeLa DAF protein revealed only minor amounts of the hydroxylamine-sensitive phospholipid species. Similar results were obtained with 125I-surface-labeled DAF from polymorphonuclear cells, as well as from unstimulated peripheral blood and anti-CD3-activated lymphocytes. These findings demonstrate that, rather than PI, the E DAF anchor contains an inositol alkylacylglycerol-phospholipid which is heterogeneous with respect to acyl groups and inositol isomers, that an ester-linked substitution in this inositolphospholipid underlies the resistance of E DAF protein to PI-PLC cleavage/release, and that this structural modification is cell-specific.

Original languageEnglish (US)
Pages (from-to)1030-1036
Number of pages7
JournalJournal of Immunology
Volume144
Issue number3
StatePublished - Jan 1 1990

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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    Walter, E. I., Roberts, W. L., Rosenberry, T. L., Ratnoff, W. D., & Medof, M. E. (1990). Structural basis for variations in the sensitivity of human decay accelerating factor to phosphatidylinositol-specific phospholipase C cleavage. Journal of Immunology, 144(3), 1030-1036.