Implications of lipid moiety in oligomerization and immunoreactivities of GPI-anchored proteins

Abstract

Glycosylphosphatidylinositol (GPI) enriches GPI-anchored proteins (GPI-AP) in lipid rafts by intimate interaction of its lipid moiety with sphingolipids and cholesterol. In addition to such lipid-lipid interactions, it has been reported that GPI may interact with protein moiety linked to GPI and affect protein conformations because GPI delipidation reduced immunoreactivities of protein. Here, we report that GPI-APs that have not undergone fatty acid remodeling exhibit reduced immunoreactivities in Western blotting, similar to delipidated proteins, compared with normal remodeled GPI-APs. In contrast, immunostaining in flow cytometry and immunoprecipitation did not show significant differences between remodeled and unremodeled GPI-APs. Moreover, detection with premixed primary/secondary antibody complexes or Fab fragments eliminated this difference in Western blotting. These results indicate that normally remodeled GPI enhanced oligomerization of GPI-APs and that inefficient oligomerization of unremodeled GPI-APs was responsible for reduced immunoreactivities. Moreover, the reduction in immunoreactivities of delipidated GPI-APs was most likely caused by the same effect. Finally, by chemical cross-linking of surface proteins in living cells and cell killing assay using a pore-forming bacterial toxin, we showed that enhanced oligomerization by GPI-remodeling occurs under a physiological membrane environment. Thus, this study clarifies the significance of GPI fatty acid remodeling in oligomerization of GPI-APs and provides useful information for technical studies of these cell components.

Fig. 1.

Processing of GPI-anchor by PGAP3 and PI-PLC. GPI-APs are subjected to fatty acid remodeling in the Golgi. An sn-2 unsaturated fatty acid (for example, C20:4) is cleaved by PGAP3 and substituted with a saturated chain (typically C18:0) by an unknown acyltransferase, remodeling GPI to two saturated long acyl chains. PI-PLC cleaves between phosphate and diacylglycerol in the phosphatidylinositol portion of GPI. The cleaved form becomes a soluble delipidated protein.

Fig. 2.

Discrepancies in GPI-APs expression between flow cytometry and immunoblotting in PGAP3-deficient cells. A: PGAP3-deficient mouse embryonic fibroblast (PGAP3^−/− MEF) cells, stably expressing either HA-PLAP, CD59, or EGFP-Flag-CD59, were infected with retrovirus vector-based mock (Vec) or PGAP3 (PGAP3) virions; GPI-AP expressions analyzed by Western blotting using whole-cell lysate or flow cytometry (left and right panels, respectively). Geometric means of surface expressions in flow cytometry are indicated in the right side as relative percentages when those in wild-type cells are presumed as 100%. B: Wild-type parent (3B2A) and derivative PGAP2/PGAP3 DM CHO cells stably expressing human CD59 and DAF were analyzed as in (A). Gray shadows, staining with indicated antibodies; dotted lines, control staining with isotype-matching antibodies; Syntaxin-6 and Ribophorin-1, quantitative controls; loading samples for Western blotting prepared under nonreducing boiling conditions.

Fig. 3.

Estimation of intracellular pools of GPI-APs. Intracellular GPI-AP pools estimated in MEF (A and B) and CHO (C and D) cells. A: Removal efficiencies of surface GPI-APs confirmed by flow cytometry. MEF cells (see Fig. 2) were incubated with PI-PLC at 10°C for 6 h or at 37°C for 30 min to prevent protein trafficking, then stained with anti-PLAP antibody. B: After cleavage of surface GPI-APs by PI-PLC, loading samples were prepared from whole-cell lysates under nonreducing boiling conditions, and intracellular HA-PLAP pools were estimated by Western blotting with anti-PLAP antibody. C and D: Wild-type parent (GD3S-C37) and derivative PGAP2/PGAP3 DM CHO cells stably expressing CD59 and DAF were treated with PI-PLC as in (A) and stained with antibodies against DAF, uPAR, and CD59 (C); loading samples were prepared as in (B), and intracellular GPI-AP pools were estimated by Western blotting using respective antibodies (D). Gray shadows, PI-PLC-treated cells; solid lines, PI-PLC-untreated cells; dotted lines, staining with isotype-matching control antibody; Syntaxin-6 and Caveolin-1, quantitative controls.

Fig. 4.

Immunoblotting with anti-GPI-AP antibodies versus immunoblotting with anti-HA antibody, flow cytometry, and enzymatic activities. A: MEF cells (see Fig. 2) were lysed, and whole-cell lysates prepared under nonreducing boiling conditions were applied to SDS-PAGE. EGFP fluorescence in gel and on transferred membrane (left and middle panels, respectively) were detected using fluorescence laser scanner; blotting membrane was then probed against 5H8 anti-CD59 antibody (right panel); nitrocellulose membrane was used instead of PVDF membrane due to high fluorescent background. The same MEF cells were used for flow cytometry, Western blotting, and enzyme assay (B–D, respectively). B: Cells were analyzed with anti-PLAP antibody in flow cytometry. Gray shadows, staining with 8B6 antibody; dotted lines, staining with isotype-matching control antibody. Numbers indicate the ratio of the geomeans. C: Loading samples were prepared from whole-cell lysates under nonreducing boiling conditions. HA-PLAP were detected with 8B6 anti-PLAP and HA7 anti-HA antibodies in Western blotting. GAPDH, quantitative control. D: ALP activities of HA-PLAP in whole-cell lysates were measured.

Fig. 5.

Conformational change-independent reduction of immunoreactivities in unremodeled GPI-APs. A: Comparison of ALP activities in PGAP3^−/− MEF cells restored with PGAP3 or mock (gray and dark gray bars, respectively) after denaturation or renaturation (left and right panels, respectively). Whole-cell lysates were prepared using Triton lysis buffer (100 mM Tris-Cl, pH 9.5, 100 mM NaCl, 5 mM MgCl₂, and 1% Triton X-100). For denaturation, SDS was added to lysate at indicated SDS percentage; for renaturation, whole-cell lysates were prepared with Triton lysis buffer containing 2.5% of SDS, then diluted to indicated SDS percentage with Triton lysis buffer without SDS. Activities were measured with 0.25 mM of CSPD, an ALP substrate. B–D: Loading samples prepared from whole-cell lysates under nonboiling (on ice, 15 min) or boiling (95°C, 3 min) nonreducing conditions; immunoblotting with anti-HA (B) and anti-PLAP (C) antibodies. C: Short exposure (bottom panel) and long exposure (top panel). D: Blotted PVDF membrane was incubated with CDP-star to measure ALP activities. Single asterisk represents partially denatured monomeric HA-PLAP due to the lack of boiling. This incomplete denaturation is most likely responsible for the broad bands. Double asterisks represent completely denatured monomeric HA-PLAP (also shown in Figs. 2–4). Triple asterisks represent enzymatically active dimeric HA-PLAP. The absolute amount of dimeric HA-PLAP was much less than monomer; therefore, HA7 anti-HA antibody barely detected the dimeric form (B), whereas anti-PLAP antibody detected the dimeric form much more strongly than monomer, most likely due to high avidity (not affinity) by divalent conjugation (C). E: Surface expression of HA-PLAP detected by anti-HA antibody in PGAP3^−/− MEF cells restored with PGAP3 or mock. Gray shadows, staining with HA7 antibody; dotted lines, staining with isotype-matching control antibody. Numbers indicate the ratio of the geomeans. F: Loading samples were prepared from whole-cell lysates of MEFs under nonreducing, nonboiling conditions, and then fluorescence intensities in SDS-PAGE were measured using fluorescence laser scanner (top panel); EGFP-Flag-CD59 was immunoblotted by anti-EGFP, anti-Flag, and anti-CD59 antibodies, and then cell lysates in (B–E) were prepared using OβG buffer.

Fig. 6.

Restoration of immunoreactivities of unremodeled GPI-APs by premixed antibody complexes. A: Immunoblotting against HA-PLAP by sequential incubations with primary and secondary antibodies (left) or by single-step incubation with premixed primary/secondary antibody complexes (right). Signals from two membranes were measured under same conditions. B: Samples from three independent experiments analyzed by sequential and single-step incubation methods (top and middle, respectively) as described in (A) and Syntaxin6, quantitative control (bottom). C: Thy-1 was immunoblotted by two methods as described in (A) (first and second panels from top) and by peroxidase-conjugated Fab fragments of anti-Thy-1 antibody (third panel from top); and Ribophorin1, quantitative control (bottom panel). D: EGFP-Flag-CD59 immunoblotted by two methods as described in (A) (top and middle panels) using anti-CD59 antibody. Ribophorin1, quantitative control (bottom panel). For single-step incubation, the same amounts of 1:1000 diluted primary and secondary antibodies were premixed at room temperature for 2 h; loading samples were prepared from whole-cell lysates under boiling (A and B) and nonboiling (C and D), nonreducing conditions.

Fig. 7.

Immunoblotting-specific reduction of immunoreactivities in delipidated GPI-APs. Whole-cell lysates prepared from MEF cells (see Fig. 2) were incubated with or without 1 unit/ml of PI-PLC from B. cereus prior to following experiments. A: Loading samples were prepared from whole-cell lysates under nonreducing boiling conditions. HA-PLAP was detected by anti-PLAP and anti-HA antibodies (top and bottom panels, respectively). B: ALP activities were measured in whole-cell lysates. C: Immunoprecipitation of HA-PLAP. Input (top and middle panels, 4% of total) and output (bottom panel, 33% of precipitates) obtained after immunoprecipitation. Proteins were immunoprecipitated from whole-cell lysates with 8B6 anti-PLAP antibody followed by protein G beads, then probed with HA7 and anti-GAPDH antibody as a control. Loading samples were prepared under nonreducing boiling conditions. The ratios of band intensities in bottom panel (output) to those in top panel (input) are shown at the bottom. D: ALP activity and immunoblotting of HA-PLAP. Loading samples were prepared from whole-cell lysates under nonreducing, nonboiling conditions. ALP activity (left), immunoblotting with anti-PLAP antibody (middle), and immunoblotting of anti-HA antibody (right). Note that the intense bands in the left and middle panels represent dimeric functional HA-PLAPs, whereas the bands in the right panel represent partially denatured monomeric HA-PLAPs, as explained in the text and Fig. 5 legend. E and F: Immunoblotting of whole-cell lysates by sequential and single-step incubations as described in Fig. 6A (left and right, respectively) with anti-PLAP (E) and anti-Thy-1 (F) antibodies. E: Single asterisk, nonspecific bands (upper); double asterisks, specific bands (lower). F: Long exposure (top) and short exposure (bottom). Note that Thy-1 is an endogenous protein. G: EGFP-Flag-CD59 was immunoprecipitated with 5H8 anti-CD59 antibody plus protein G beads. Loading samples (8% of total lysates for input and 40% of the precipitates for output) were prepared under nonreducing, nonboiling conditions and applied to SDS-PAGE. Fluorescence was measured by fluorescence laser scanner, and the ratios of band intensities in output to those in input were calculated and are shown at the bottom. H: Immunoreactivity of EGFP-Flag-CD59 after incubation with or without PI-PLC was analyzed as described in Fig. 5F.

Fig. 8.

Fatty acid remodeling accelerates oligomerization of GPI-APs under physiological membrane environments. PGAP3^−/− MEF cells restored with PGAP3 or mock that expressed HA-PLAP (A and B) and HA-Thy1 (C) were suspended in lysis buffer containing 1% TritonX-100, and then separated into detergent-resistant membrane (DRM) fraction representing lipid rafts and detergent-soluble (Soluble) fraction representing nonrafts by low-speed centrifugation (A and C) or by sucrose density-gradient ultracentrifugation (B). A: HA-PLAP expressed was detected by anti-PLAP antibody (third panel from top) and anti-HA antibody (bottom panel). B: HA-PLAP in each fraction after ultracentrifugation was detected by HA7 anti-HA antibody. C: Thy1 was detected using premixed G7 anti-Thy-1 and HRP-conjugated anti-Rat IgG antibodies complex (bottom panel). Loading samples prepared under nonreducing boiling conditions. Cav1, caveolin1; TfR, transferrin receptor; Flotillin and caveolin1 were used as detergent-resistant membrane (DRM) markers, and transferrin receptor was used as detergent-soluble fraction marker. D: Cells expressing HA-Thy1 were treated with 1 mM of cross-linking reagent DTSSP for 30 min at room temperature. Loading samples were prepared from whole-cell lysate under nonreducing, nonboiling conditions. Three independent experiments are shown. Single and double asterisks indicate monomeric and dimeric HA-Thy1 or endogenous Thy1; long exposure (top); short exposure (bottom). E: The ratios of dimers to monomers were quantitatively calculated. Statistical analysis was done with Student-t-test (P = 0.014). F: Cells were stained with 10⁻⁸ M or 10⁻⁹ M of nontoxic FITC-conjugated proaerolysin (FLAER). Bold line, PGAP3-restored PGAP3^−/− MEF cells; continuous line, empty vector integrated PGAP3^−/− MEF cells; dotted line, no staining cells as a control. G: Viabilities of MEFs after 24 h cultivation with fresh medium following treatment with indicated concentrations of aerolysin (nM) for 3 h. The cell number at starting point was regarded as 100%. PGAP3, PGAP3-restored PGAP3^−/− MEF cells; Vec, empty vector integrated PGAP3^−/− MEF cells.