Structural and functional characterization of the inositol phospholipid of decay accelerating factor's glycolipid anchor
The glyco-inositol phospholipid (GIPL) anchoring moieties of decay accelerating factor (DAF) proteins expressed on human erythrocytes (E hu) and nucleated cells were structurally compared to determine the mechanism of their differing sensitivity to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC). Gas chromatographic (GC) analyses of methanolyzed, silylated, and hydrolyzed/acetylated E hu DAF protein revealed 2.20 ± 0.16 mol of fatty acid, 0.86 ± 0.05 mol of inositol, and ?1.0 mol of alkylglycerol per mol of protein. Nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) revealed that pretreatment of the PI-PLC-resistant E hu DAF with alkaline hydroxylamine, a reagent which removes ester- but not ether-linked lipids, rendered it PI-PLC susceptible. Parallel analyses of nucleated cell DAF revealed only minor amounts of the hydroxylamine-sensitive phospholipid species and a predominance of alkylacylglycerol. These findings demonstrate that DAF's anchor phospholipid is an inositol alkylacylglycerol, and that an ester-linked substitution of the inositol underlies the resistance of E hu DAF to PI-PLC. To determine whether acylation of anchor inosito l is protein- or cell-specific, two K562 cell lines (ATCC and the subclone K562.48), which exhibit alternatively PI-PLC-sensitive and -resistant endogenous DAF proteins, were transfected with placental alkaline phosphatase (PLAP) cDNA. The extent inositol acylation in the exogenous PLAP and endogenous DAF proteins was compared. ND-PAGE analyses showed that in K562.48 cells, like the endogenous DAF, there was >95% PLAP molecules with acylated inositol. In K562-ATCC cells there were populations of both DAF and PLAP with acylated and unacylated inositol, but the proportions of these populations differed between the two proteins. These results indicate that GIPL anchor inositol acylation is regulated in a cell specific fashion but that certain proteins properties may play a role in the percentage of each anchor type expressed. To assess the effect of the inositol-associated fatty acid and of the glycerol sn-2 acyl substituent on DAF's functional efficiency, endogenous E hu and HeLa DAF proteins, bearing three- and two-"footed" GIPL anchors, and one-"footed" synthetic DAF variants retaining either glycerol- or inositol-associated anchor lipids were functionally compared. All four anchor variants bound to cells and inhibited expression of C4b2a hemolytic activity. However, only the endogenous (E hu and HeLa) proteins (which exhibited similar functional efficiency) reinserted into the lipid bilayer and functioned intrinsically. These findings indicate that an intact plasmanylinositol-containing anchor is necessary for membrane incorporation and intrinsic DAF function and that inositol acylation has no effect on DAF function.
School:Case Western Reserve University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:structural inositol phospholipid glycolipid anchor
Date of Publication:01/01/1991