Lipid modification of proteins through sortase-catalyzed transpeptidation

Abstract

A general chemoenzymatic method for the site-specific attachment of lipids to protein substrates is described. Sortase A is used to append short lipid-modified oligoglycine peptides to the C terminus of protein substrates bearing a five amino acid sortase A recognition sequence (LPETG). We demonstrate the attachment of a range of hydrophobic modifications in excellent yield (60-90%), including a simple step for removing the sortase enzyme postreaction. Lipoproteins prepared using these procedures were subsequently shown to associate with mammalian cells in a lipid tail-dependent fashion and localized to the plasma membrane and endosomes.

Figure 1

(a) Optimization of a two step protocol for lipid ligation followed by removal of the sortase enzyme. Conditions: 77 µM eGFP-LPETG-His₆, 150 µM sortase A, 2 mM nucleophile, 1% (w/v) detergent, 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl₂, 3 h at 37 °C. After transpeptidation, Ni-NTA resin was added as a slurry in 1 M NaCl and 40 mM imidazole and incubated for 2 h at RT. Abbreviations: OG = n-octyl glucoside, DM = n-dodecyl maltoside, DOC = deoxycholate. *Samples not treated with Ni-NTA resin. (b) ESI-MS spectrum of eGFP-LPET-1-C22 ligation product.

Figure 2

Lipid attachment through sortase-catalyzed transpeptidation. (a) Optimized procedure for lipid coupling followed by removal of His₆-tagged proteins with Ni-NTA resin. (b) SDS-PAGE analysis of lipid-modified eGFP following transpeptidation and depletion of His₆-tagged proteins. [Input = eGFP-LPETG-His₆ in the absence of sortase and nucleophile, *Samples incubated with Ni-NTA resin and 300 mM imidazole to block binding of His₆-tagged proteins].

Figure 3

Association of lipid-modified eGFP with HeLa cells. Cells were incubated with lipid-modified eGFP (2.5 µg/mL) for 1 h in serum-free medium and then analyzed by flow cytometry. Numbers in parentheses represent the fold enhancement in mean cellular fluorescence relative to eGFP-GGG. Histograms for eGFP-GGG (black) and eGFP-1-ad (gray) are overlapping.

Figure 4

Subcellular distribution of eGFP-1-C22 and eGFP-2-chol in U373 and HeLa cells. Cells were incubated with lipid-modified eGFP (2.5 µg/mL) at 37 °C in serum-free medium and then imaged live using spinning disc confocal microscopy. (a) U373 cells 1 h incubation, (b) U373 cells 5 h incubation, (c) HeLa cells 1.25 h incubation, (d) HeLa cells 5 h incubation.

Figure 5

eGFP-1-C22 is able to access early endosomal compartments as determined by partial colocalization with Transferrin-Alexa 647. Cells were incubated with eGFP-1-C22 (2.5 µg/mL) for 5 h in serum-free medium followed by the addition of Transferrin-Alexa 647 (100 µg/mL) during the final 15 minutes. Significant colocalization between eGFP-2-chol and Transferrin-Alexa 647 was not observed.

Scheme 1

Site-specific lipid attachment through sortase-mediated transpeptidation.

Scheme 2

Synthesis of lipid-modified triglycine nucleophiles.