Rescue of Glycosylphosphatidylinositol-Anchored Protein Biosynthesis Using Synthetic Glycosylphosphatidylinositol Oligosaccharides

The attachment of proteins to the cell membrane using a glycosylphosphatidylinositol (GPI) anchor is a ubiquitous process in eukaryotic cells. Deficiencies in the biosynthesis of GPIs and the concomitant production of GPI-anchored proteins lead to a series of rare and complicated disorders associated with inherited GPI deficiencies (IGDs) in humans. Currently, there is no treatment for patients suffering from IGDs. Here, we report the design, synthesis, and use of GPI fragments to rescue the biosynthesis of GPI-anchored proteins (GPI-APs) caused by mutation in genes involved in the assembly of GPI-glycolipids in cells. We demonstrated that the synthetic fragments GlcNAc-PI (1), Man-GlcN-PI (5), and GlcN-PI with two (3) and three lipid chains (4) rescue the deletion of the GPI biosynthesis in cells devoid of the PIGA, PIGL, and PIGW genes in vitro. The compounds allowed for concentration-dependent recovery of GPI biosynthesis and were highly active on the cytoplasmic face of the endoplasmic reticulum membrane. These synthetic molecules are leads for the development of treatments for IGDs and tools to study GPI-AP biosynthesis.

1 H, 13 C and 31 P-NMR as well as 2D-spectra (COSY, HSQC and HMBC) were recorded on a Bruker 400 (400 MHz) or a Bruker Ascend 400 (400 MHz) spectrometers; the chemical shifts are expressed in parts per million (pmm) referenced to solvent signals. Data for 1 H RMN are reported as follows: δ, chemical shift; multiplicity (recorded as br, broad; app, apparent; s, singlet; d, doublet; t, triplet; q, quadruplet; and m, multiplet), coupling constants (J in Hertz,
After stirring for 2 h at room temperature, the reaction was quenched with Et3N and concentrated under reduced pressure. The obtained residue was purified by flash silica gel column chromatography to afford the corresponding pseudo-disaccharide as a 7:1 α/β mixture that could not be separated; Rf = 0.34 (hexane/EtOAc, 7:3). The product was dissolved in an 11:1 MeOH/DCM anhydrous mixture and 61 µL (0.121 mmol) of a 5M NaOMe solution in MeOH was added. The reaction was stirred at 40 °C for 1 h before it was neutralized with Amberlite IR 120 (H + ) resin, filtered and concentrated in vacuo. The resulting residue was purified by flash silica gel column chromatography to give the desired triol in 77% yield (29.6 mg, 0.0312 mmol) as a white solid. The α and β anomers were separated during the column; Rf α-anomer= 0.40, Rf β-anomer= 0.27 (hexane/EtOAc, 3:7). NaH (20.3 mg, 0.509 mmol, 60% in mineral oil) was added to a solution of the isolated αanomer (80.4 g, 0.0848 mmol) and TBAI (141 mg, 0.382 mmol) in DMF (1.2 mL) at 0 °C. After 20 min, 2-(Bromomethyl)naphthalene (84.4 mg, 0.382 mmol) was added, and the reaction mixture was stirred at room temperature for 2 days. The reaction was quenched with water, and volatiles were removed under reduced pressure. The remaining was dissolved with ethyl acetate, and the solution was extracted with water. The layers were separated, and the aqueous phase was extracted with ethyl acetate. Organic layers were combined, washed with brine, dried with Na2SO4, filtered, and evaporated. The resulting residue was purified by flash silica gel column chromatography to give 16 in 76% (88.2 mg, 0.0644 mmol) yield as a white solid; Rf = 0.34 (hexane/EtOAc,8:2).
Compound treatment of cells. Dried compounds were reconstituted with DMSO at a concentration of 10 mM (stock solution). Cells (1x10 5 /well) were incubated in 12 well plates overnight. The medium was removed and 500 μL of serum free medium (D-MEM/Ham's F-12) containing the described concentrations of compounds was added. After 24 hours incubation, cells were analyzed by FACS. For further PIPLC treatment, compound treated cells were harvested and incubated in 50 μL of serum free medium containing 0.05 U/mL of PIPLC (Thermo Fisher Scientific) for 1.5 h at 37°C and were analyzed by FACS.
Pulse-chase analysis of treated cells. PIGA-KO and PIGL-KO cells were incubated in 24 well plates with 5x10 4 cells per well for overnight and pulsed with 250 μL of serum free medium containing 10 μM of compounds for 1h at 4°C. After washing, the cells were chased and analyzed by FACS. Percent restorations of CD59 expression to wild type cells were plotted in various time points.

Rescue experiments using Streptolysin O.
Cells were incubated in HBSS with 130 μg/mL of streptolysin O (Bioacademia) on ice for 5 min and were washed with PBS. Then, cells were incubated with compounds or streptoavidin-alexa488 for a positive control in 250 µL of HBSS (20 μM final concentration for 4 and 5, 2 μM for GlcNAcPI 1, or 0.5 μg SA-Alexa488), for 15 min, at 37 °C. Following, 750 µL of serum free medium containing CaCl2 were added to seal the holes made by streptolysin O. The cells were incubated for 24 h and were analyzed to determine CD59 expression by FACS.