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.

Keywords: GPI anchor; glycoproteins; glypiation; protein modifications; protein semisynthesis.

Figure 1

a) Lipid and glycan variability of GPIs anchoring Plasmodium spp. proteins. b) Structure of the cysteine‐containing GPIs and biotin used in this study.

Figure 2

One‐pot ligation of eGFP with biotin. a) Scheme of the OPL reaction. b) Comparison of OPL with MESNA, MMP, and MMBA by SDS‐PAGE and western blot (eGFP‐Biotin detected with anti‐biotin antibody). M=Molecular weight marker; 1) GFP‐Npu ^N; 2) OPL with MESNa, 1 h; 3) OPL with MMP, 1 h; 4) OPL with MMBA, 1 h; 5) OPL with MESNa, 1 d; 6) OPL with MMP, 1 d; 7) OPL with MMBA, 1 d. c) Kinetic study of OPL, monitored by RP‐HPLC (C4). d) SDS‐PAGE and western blot of the OPL of eGFP with GPI2; detection with Ponceau S staining and anti‐GPI antibody after 1,2 and 6 days. M=Molecular weight marker, EP=eGFP‐Npu ^N. e) SDS‐PAGE of OPL between Thy1 and GPI1; purification by His‐Trap. M=Marker; LM=ligation mixture 6 d; FT=flow‐through; W=wash; E=elution fractions. MESNa=sodium 2‐mercaptoethanesulfonate, MMP=methyl 3‐mercaptopropionate.

Scheme 1

Synthesis of lipidated GPIs containing a cysteine residue. Conditions and reagents: a) PdCl₂, AcOH, H₂O, AcONa, 70 %. b) i. 7,16 PivCl, pyridine; ii. I₂, H₂O 75 %. c) Sc(OTf)₃, H₂O, CH₂Cl₂/acetonitrile, 68 %. d) i. 10,13b PivCl, pyridine; ii. I₂, H₂O, 70 %. e) i. H₂N‐NH₂‐AcOH, AcOH, pyridine, CH₂Cl₂; ii. Pd(OH)₂/C, H₂, CHCl₃, MeOH, H₂O; iii. TFA, anisole, Hg(OTFA)₂, 0 °C, iv. β‐mercaptoethanol, H₂O, 43 % (over 4 steps). Piv=pivaloyl, TfO=trifluoromethanesulfonate, TFA=trifluoroacetic acid.

Figure 3

Characterization of GPI‐anchored proteins. a) RP‐HPLC chromatogram of purified MSP1₁₉‐GPI3. b) Deconvoluted ESI‐MS spectrum of MSP1₁₉‐GPI3. c) SDS‐PAGE after His‐Trap purification of the preparative synthesis of MSP1₁₉‐Biotin and MSP1₁₉‐GPI3. M=molecular weight standard, FT=flow through; EP=MSP1₁₉‐Npu ^N, LM=ligation mixture; E=elution fractions; dotted arrow shows the intein. d) CD spectra of MSP1₁₉‐Biotin and MSP1₁₉‐GPI3, e, f) Production of TNF‐α (e) and IL‐12 (f) by BMDCs stimulated with the MSP1₁₉‐Biotin and MSP1₁₉‐GPI3. CM=Culture medium.