Abstract
The inositol ring in the glycoinositolphospholipid (GPI) anchor of human decay-accelerating factor (DAF) is unmodified in nucleated cells, whereas it is fatty acid acylated in erythrocytes (Ehu). To assess the effect of this and of the glycerol sn-2-associated acyl substituent on the abilities of DAF to cell membrane incorporate and function, 1) endogenous (physiologically anchored) DAF proteins bearing three- and two-"footed" GPI anchors were purified from Ehu and HeLa cells and 2) synthetic DAF variants bearing alternative one- "footed" anchors (retaining either the sn-1 glycerol- or inositol-associated lipid) were prepared by alkaline hydroxylamine treatment and phosphatidylinositol-specific phospholipase D digestion of Ehu DAF, respectively. The different DAF species were added to antibody-sensitized sheep erythrocytes (EshA) and their abilities to insert into the plasma membranes of the cells and control subsequent complement activation on their surfaces were compared. DAF proteins bearing all four GPI anchor structures adhered to the Esh hemolytic intermediates and inhibited expression of C3 convertase (C4b2a) activity. However, mixing of DAF-treated EshA with untreated EshAC142 and stripping of cell-associated DAF proteins with vesicles showed that only the physiologically anchored proteins remained stably associated with the lipid bilayer and functioned intrinsically. Both three- and two-"footed" Ehu and HeLa DAF proteins exhibited comparable ability to incorporate and function in the intermediates as well as to accumulate to levels 1000-fold higher/cell in Schistosoma mansoni schistosomula. These findings indicate that 1) an intact inositolphospholipid-containing GPI anchor is necessary for stable membrane integration and intrinsic function, 2) endogenous GPI anchors (with either unsubstituted and acylated inositol) incorporate and function with comparable efficiency, and 3) the transfer of either endogenous DAF form can account for the previously described circumvented uptake of human C3b by blood stage schistosomula.
Highlights
The inositol ring in the glycoinositolphospholipid Decay-accelerating factor (DAF),’ a 70-kDa cell surface (GPI) anchor of human decay-accelerating factor protein, is a potent inhibitor of complement activation which (DAF) is unmodified in nucleated cells, whereas i t is serves to protect host tissues from autologous complement fatty acid acylated in erythrocytes (E””).To assess the attack
The differentDAF species were added to the inositolphospholipid within it renders the isolated protein antibody-sensitized sheep erythrocytes (E””A) and able to reintegrate into plasma membranes when added to their abilities to insert into theplasma membranes of cells in uitro [10]
The comparisons between EhUand HeLa cell DAF function shown), of DAF molecules with both acylated and unacylated carried out in the present study demonstrattehat thepresence GPI anchor inositol, demonstrates that thereis no selectivity in theincorporation processbased on inositol acylation
Summary
0.08% Triton X-100, the mixture wasdialyzed extensively against phosphate-buffered saline containing 0.08% Triton X-100. M Tris, pH 7.4, 10 mMMgC1, and 2 mM CaCb. Alkylglycerol has not seen directly measured in nuclealed cell For preparation of alkylglycerolDAF, 50 p1 of the labeled Eh"DAF proteins with GPI anchorsbut inferred from ND-PAGE analyses. Preparation (in 0.1% Triton X-100 phosphate-buffered saline) was incubated overnight a t 0 "C with 200 pl of 1.0 M hydroxylamine, 0.1 M triethylamine, pH 10.7. For their preparation, DMPC was suspended in buffer a t 20 mM phospholipid concentration and thesuspension sonicated for 50 were not released from alkylacylglycerol to leave the single inositol-associated lipid which would still bind to the membrane), cells were hypotonically lysed and after collection of stroma by centrifugation a t 100,000 X g, the associated 1251DAF was extracted from the membranes with 1%Triton X-.
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