Semisynthesis of Functional Glycosylphosphatidylinositol‐Anchored Proteins

Abstract Glypiation is a common posttranslational modification of eukaryotic proteins involving the attachment of a glycosylphosphatidylinositol (GPI) glycolipid. GPIs contain a conserved phosphoglycan that is modified in a cell‐ and tissue‐specific manner. GPI complexity suggests roles in biological processes and effects on the attached protein, but the difficulties to get homogeneous material have hindered studies. We disclose a one‐pot intein‐mediated ligation (OPL) to obtain GPI‐anchored proteins. The strategy enables the glypiation of folded and denatured proteins with a natural linkage to the glycolipid. Using the strategy, glypiated eGFP, Thy1, and the Plasmodium berghei protein MSP119 were prepared. Glypiation did not alter the structure of eGFP and MSP119 proteins in solution, but it induced a strong pro‐inflammatory response in vitro. The strategy provides access to glypiated proteins to elucidate the activity of this modification and for use as vaccine candidates against parasitic infections.


Synthesis of the Npu C Peptide
Npu C AA -Sequence: IKIATRKYLGKQNVYDIGVERDHNFALKNGFIASAAFNA The modified C-terminal intein Npu C AA was synthesized via solid phase peptide synthesis (SPPS) using a microwave peptide synthesizer (Liberty Blue, CEM, USA). [1] Rink amide TentaGel S RAM resin was swollen in dichloromethane (DCM) and transferred to the reaction vessel of the synthesizer. The synthesis was run on the synthesizer using 1 M ethyl cyano(hydroxyimino)acetate (Oxyma) [2] with 0.1 M N,N-diisopropylethylamine (DIPEA) in dimethylformamide (DMF) as activator base and 0.5 M N,N′-diisopropylcarbodiimide (DIC) in DMF as activator. The fluorenylmehtyloxycarbonyl (Fmoc) group was removed after each coupling step by treatment twice with 20% piperidine in DMF containing 1% formic acid. [3] The peptide was cleaved from the resin with trifluoroacetic acid (TFA) containing triisopropylsilyl (TIPS) in water (90:5:5) by shaking the reaction for 2 h at room temperature. The cleaved peptide was precipitated and washed with ice-cold ether three times. Peptides were analyzed by RP-HPLC on a C18 Hydrosphere column (3 x 50 mm, YMC, Japan) using a 30minute gradient of 5 -70% acetonitrile in water with 0.1% TFA. The products were analyzed by MALDI-MS using 2,3-dihydroxybenzoic acid (DHB) as matrix and a reflector method in positive ion mode. The peptides were purified using a Waters HPLC system with a Hydrosphere C18 semi-preparative column (10 x 150 mm, YMC, Japan). Fractions containing the product were collected and lyophilized to give 20 mg of the Npu C AA. The isolated product was analyzed by LC-MS and MALDI-TOF-MS.

RP-HPLC Analysis for Peptide Samples.
Peptide samples were analyzed in RP-HPLC using a Hydrosphere C18 column (50 x 3 mm, YMC, Japan) in an 1100 system (Agilent, USA), using a gradient from 5 -70% acetonitrile in water (0.1% TFA) in 30 min. Chromatograms were analyzed using ChemStation software.

Expression and characterization of Thy1-Npu N
Plasmid pET30a-Thy1-NpuN was ordered from GenScript (Piscataway, USA) and transformed into E. coli BL21 Rosetta cells using heat shock transformation and chemically competent cells. 1 L LB medium were inoculated to OD600 = 0.05, grown to late exponential phase (OD600 of 0.6 -0.8) at 37°C, 220 rpm and protein expression was induced by addition of 0.5 mM IPTG. The temperature was changed to 30°C and shaking speed was reduced to 180 rpm. After 24 h induction, the cells were harvested by centrifugation (4.000 x g, 10 min, 4°C), the pellet was resuspended in 25 mL lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 10 mM Imidazole) + 0.25 mg/mL lysozyme + protease inhibitors and incubated for 1 h at room temperature, 120 rpm. The suspension was subsequently sonicated for 20 min (pulses of 30 sec, 20% amplitude, on ice) and centrifuged (12.000 x g, 1 h, 4°C). The fusion protein was expressed in insoluble form, the lysis was repeated to remove all soluble proteins. The supernatant was discarded and the inclusion body pellet was solubilized in 20 mL HisTrap equilibration buffer + 8 M urea by vortexing, ultrasonic bath incubation for 2 h at room temperature (120 rpm). The solubilized inclusion bodies were centrifuged for 1.5 h (24.400 x g, 4°C) and filtered through a 0.2 µm syringe filter prior HisTag purification under denaturing conditions (8 M urea). For this, 8 mL lysate per run were loaded onto a HisTrap FF (5 mL) column equilibrated in HisTrap equilibration buffer + 8 M urea (on Knauer Azura FPLC, flow rate 1 mL/min). The column was washed with equilibration buffer + 8 M urea for 10 CV following elution of unbound host proteins, and the fusion protein was eluted using a gradient from 30 mM to 1 M imidazole over 5 CV. The fractions were analyzed in SDS-PAGE and fractions containing the fusion protein were combined and dialyzed into ligation buffer + 6 M urea. 40 mg fusion protein was obtained from 1 L culture volume. The purification process is shown in Figure S3.

Expression and Characterization of MSP119-Npu N
Plasmid pET29a+-MSP119-Npu N was ordered from GenScript (Piscataway, USA) and transformed into E. coli BL21 Star (DE3) using heat shock transformation and chemically competent cells. 1 L LB medium were inoculated to OD600 = 0.05, grown to late exponential phase (OD600 of 0.6 -0.8) at 37°C, 220 rpm and protein expression was induced with 0.5 mM IPTG and the temperature was changed to 25°C m. After 7 h induction, the cells were harvested by centrifugation (4.000 x g, 10 min, 4°C), the pellet was resuspended in 25 mL lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 10 mM Imidazole) + 0.25 mg/mL lysozyme + protease inhibitors and incubated for 1 h at room temperature. The suspension was subsequently sonicated for 30 min using pulses of 60 sec. and 30 sec. pause, at 37°C, and centrifuged (12.000 x g, 2 h, 4°C).
The supernatant was loaded onto a HisTrap FF (5 mL) column equilibrated with 20 mM Tris buffer (pH 8.0, 500 mM NaCl) buffer. The column was washed with 10 CV of the buffer to remove unbound proteins. The fusion protein MSP119-Npu N was eluted using a gradient from 30 mM to 1 M imidazole in 50 mM Tris buffer (pH 8.0, 150 mM NaCl) over 10 CV. The fractions were analyzed in SDS-PAGE and fractions containing the fusion protein were combined and dialyzed against 50 mM Tris buffer (pH 8.0, 10 mM NaCl). To separate proteins with higher molecular weight, the collected fractions from the HisTrap were eluted on a Superdex 75 column to obtain 6-10 mg of the MSP119-Npu N protein. Purification of MSP119-Npu N is shown in Figure S5

Optimization of OPL between Thy1-Npu N and Cys-Biotin 4.
To a solution of 15 µM fusion protein Thy1-Npu N (1 mg) and 1.2 equivalents of Npu C AA peptide in ligation buffer (20 mM Tris/HCl pH 7.0, 150 mM NaCl) containing 6 M urea, TCEP was added to a final concentration of 1, 5 and 10 mM. The cysteine Biotin 4 (10 equivalents) and the thiol reagent (50 mM of MMP) were added to the mixture. The ligation was incubated at 37 °C during six days. The reaction mixture was passed through a HisTrap column to separate the unreacted fusion protein and the cleaved Npu N fragment having a HisTag. The reaction progress and the isolation of the product was monitored using RP-HPLC, SDS-PAGE and western blotting, Figure S9.

OPL between GPI1 and Thy1-Npu N .
The OPL was completed under the same conditions as described for cysteine biotin. Only 1 equivalent of the thiol reagent, (50 µg, 0.035 µmol) GPI1, was added to the reaction.  To a solution of 13 µM fusion protein MSP119-Npu N (0.1 mg) and 1.5 equivalents of Npu C AA peptide in ligation buffer (20 mM Tris/HCl pH 7.0, 150 mM NaCl), TCEP was added to a final concentration of 0.5 mM. The cysteine-Biotin 4 (4 equivalents) and the thiol reagent (50 mM of MMBA) were added to the mixture. The ligation was incubated at 37 °C and samples were taken after 1, 14 and 24 hours. The reaction progress was monitored using SDS-PAGE and western blot.

OPL reaction between MSP119-Npu N and GPIs.
To a solution of 13 µM fusion protein MSP119-Npu N (0.1 mg) and 1.5 equivalents of Npu C AA peptide in ligation buffer (20 mM Tris/HCl pH 7.0, 150 mM NaCl), TCEP was added to a final concentration of 0.5 mM. The cysteine containing GPI1 and GPI3 (4 equivalents) and the thiol reagent (50 mM of MMBA) were added to the mixture. The ligation was incubated at 37 °C and samples were taken after 1, 24 and 72 hours. The reaction progress was monitored using SDS-PAGE and western blot. The reaction with GPI3 was performed with and without of octyl glucoside (20 mM) as detergent. The reaction mixture was separated using a HisTrap column (1 mL). The unbound protein and peptide were collected and separated by SEC on a GE Healthcare Superdex 30 (16 x 600 mm) column. The fractions containing the product were concentrated to a final volume of 1 mL using a 5 mL centrifugation tube with a 5 KDa membrane filter to give 0.405 mg of the ligation product (70 % yield).

Structural Analysis by Circular Dichroism
To evaluate the effect of the OPL reaction conditions and from the biotinylation and glypiation on the structures of the eGFP and MSP119 proteins, circular dichroism analysis was performed.
For CD measurements, protein samples were prepared in PBS buffer at concentrations ranging from 0.1 -1 mg/mL, depending on the amounts available. The protein solution was then filled into a high precision cell quartz cuvette (light path 1 mm) and placed in the Chirascan Circular Dichroism Spectrometer. Circular dichroism was measured between 195 and 250 nm against PBS buffer as a reference and measurements were performed in triplicates. The averages of these measurements were plotted over the wavelength. The β-sheet structure of eGFP is not compromised by C-terminal modification with biotin and GPI1. The protein also retains its fluorescence.

General Methods for Synthetic chemistry
All purchased chemicals were of reagent grade and all anhydrous solvents were of high-purity grade and used as supplied except where noted otherwise. Reactions were performed in ovendried glassware under an inert argon atmosphere unless noted otherwise. Reagent grade thiophene was dried over activated molecular sieves prior to use. Pyridine was distilled over CaH2 prior to use. Sodium hydride suspension was washed with hexane and THF and stored in an anhydrous environment. Benzyl bromide was passed through activated basic aluminum oxide prior to use. Metal sodium was washed with hexane and stored in hexane. Analytical thin layer chromatography (TLC) was performed on Merck silica gel 60 F254 plates (0.25mm). Compounds were visualized by UV irradiation or heating the plate after dipping in staining solution. The staining solutions were cerium sulfate-ammonium molybdate (CAM) solution, basic potassium permanganate solution, acidic ninhydrin-acetone solution, or a 3methoxyphenol-sulfuric acid solution (Sugar Stain). Flash column chromatography was carried out using a forced flow of the indicated solvent on Sigma Aldrich silica gel high purity grade 60 Å (230-400 mesh particle size, for preparative column chromatography). Solvents were removed under reduced pressure using rotary evaporator and high vacuum (<1 mbar). Freeze drying of the aqueous solutions was performed using Alpha 2-4 LD Lyophilizer (Christ, Osterode am Harz, Germany) 1 H, 13  . Splitting patterns are indicated as s, singlet; d, doublet; t, triplet; q, quartet; br, broad singlet; dd, doublet of doublets; m, multiplet; dt, doublet of triplets; h, hextet for 1 H NMR data. Signals were assigned by means of 1 H-1 H COSY, 1 H-1 H TOCSY, 1 H-13 C HSQC, 1 H-13 C HMBC spectra and version thereof. ESI mass analyses were performed by Waters Xevo G2-XS Q-TOF with an Acquity H-class UPLC and a Bruker Autoflex-speed MALDI-TOF spectrometer.

1,2-di-O-stearoyl-sn-glycero-3-H-phosphonate (7) [8]
A mixture of 1,2-distearoyl-sn-glycerol (250 mg, 0.40 mmol) and phosphonic acid (36.1 mg, 0.44 mmol) were dissolved and co-evaporated three times with pyridine. The resulting mixture was dissolved in anhydrous pyridine (10 mL) and pivaloyl chloride (0.05 mL, 0.32 mmol) was added dropwise. [9] The reaction mixture was stirred under nitrogen atmosphere at room temperature for two days. The volatiles were removed under reduced pressure and the resulting solid was purified by silica gel column chromatography to give H-phosphonate 7 (218 mg, 0.32 mmol, 79%) as a white solid.  The pseudo-pentasaccharide 6 (98 mg, 0.041 mmol) and H-phosphonate 7 (161 mg, 0.203 mmol) were dissolved and co-evaporated with anhydrous pyridine (3 x 3 mL) and dried under high vacuum overnight. The reaction mixture was dissolved in anhydrous pyridine (2 mL) at room temperature and pivaloyl chloride (20 µL, 0.163 mmol) was added. [9] The resulting reaction mixture was stirred at room temperature for 3 h. Iodine (36.3 mg, 0.143 mmol) in a mixture of pyridine/water (19:1, 0.2 mL) was added to oxidize P (III) to P (V). The reaction mixture was further stirred for 2 h at room temperature to the complete the oxidation. The reaction mixture was diluted with CHCl3 and washed with aqueous Na2S2O3 solution to remove the excess iodine. The aqueous layer was further washed with CHCl3 and the combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude residue, which was subjected to flash column with Et3N-deactivated silica gel to give 8 (70 mg, 55 %) as a syrup. Rf = 0.25 (CHCl3/MeOH = 20: 1). 1
The bilipidated pseudo-pentasaccharide 9 (40 mg, 0.014 mmol) and H-phosphonate 10 [10] (39 mg, 0.102 mmol) were dissolved and co-evaporated with anhydrous pyridine (3x3 mL) and dried under high vacuum overnight. The reaction mixture was dissolved in anhydrous pyridine (2 mL) at room temperature and pivaloyl chloride (13 µL, 0.102 mmol) was added. The resulting reaction mixture was stirred at room temperature for 5 h. The progress of the reaction was monitored by using TLC analysis. Iodine (26 mg, 0.102 mmol) in a mixture of pyridine/water (19:1, 0.2 mL) was added to oxidize P (III) to P (V). The reaction mixture was further stirred for 2 h at room temperature to complete the oxidation, diluted with CHCl3, washed with aqueous Na2S2O3 solution to remove the excess iodine. The aqueous layer was further washed with CHCl3 and the combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude residue, which was subjected to flash column with Et3N-deactivated silica gel to give 11 (32 mg, 70 %) as a syrup. 1