Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen

A variety of pathogenic bacteria infect host eukaryotic cells using protein toxins, which enter the cytosol and exert their cytotoxic effects. Anthrax lethal toxin, for example, utilizes the membrane-spanning translocase, protective antigen (PA) pore, to deliver the protein toxin lethal factor (LF) from the endosome into the cytosol of cells. Previous work has investigated the delivery of natural peptides and enzymatic domains appended to the C-terminus of the PA-binding domain of lethal factor (LFN) into the cytosol via PA pore. Here, we move beyond natural amino acids and systematically investigate the translocation of polypeptide cargo containing non-canonical amino acids and functionalities through PA pore. Our results indicate translocation is not perturbed with alterations to the peptide backbone or side-chain. Moreover, despite their structural complexity, we found that the small molecule drugs, doxorubicin and monomethyl auristatin F (MMAF) translocated efficiently through PA pore. However, we found cyclic peptides and the small molecule drug docetaxel abrogated translocation due to their large size and structural rigidity. For cargos that reached the cytosol, we demonstrated that each remained intact after translocation. These studies show PA is capable of translocating non-canonical cargo provided it is in a conformational state conducive for passage through the narrow pore.


Solid phase peptide synthesis (Boc)
Select peptides were synthesized using in situ neutralization boc chemistry. Peptides were synthesized on 0.2 mmol scale on MBHA resin and the following side chain protection was used for L-and D-amino acids: Arg(Tos), Asn(Xan), Asp(OcHex), Lys(2-ClZ), Lys(Alloc), and Ser(Bzl). For the cyclic peptides, peptide thioesters were prepared using the S-trityl mercaptopropionic acid (MPA) strategy. After peptide synthesis, the peptides were cleaved from the resin and side chains were deprotected using 10% (v/v) p-thiocresol and 10% (v/v) p-cresol in anhydrous HF for 1 h at 0 °C. Peptides were then triturated with cold diethyl ether, dissolved in 50:50 A:B (A: water + 0.1% TFA and B: acetonitrile + 0.1% TFA), and then lyophilized.

Synthesis of docetaxel-maleimide
Docetaxel (100 mg, 0.124 mmol) and maleimidopropionic acid (25 mg, 0.149 mmol) were taken in anhydrous DCM (1.5 mL), followed by addition of Mukaiyama's reagent; 2-chloro-1methylpyridinium iodide (57 mg, 0.225 mmol) and excess triethylamine (0.2 mL) at 0°C. The reaction mixture was slowly warmed up to room temperature and stirred 16 hours, at which TLC analysis (5% v/v methanol in dichloromethane) indicated consumption of starting materials and formation of a major product. The reaction was quenched by addition of ethanol and additional stirring for 10 min, followed by concentration to dryness. The crude material was subjected to silica flash chromatography to give the thiol reactive docetaxel derivative, docetaxel-maleimide, 62 mg (52.2 % yield). The identity of the product was confirmed by high resolution LCMS and 1 H-NMR (supplementary information).

Synthesis of doxorubicin-maleimide
Doxorubicin (50 mg, 0.086 mmol) and N-succinimidyl ester of maleimidopropionic acid (45.78 mg, 0.172 mmol) were taken in DMF (1.6 mL) and reacted for 1 hour in the presence of N,Ndiisopropylethylamine DIEA (50 µL), at which TLC analysis (20% v/v methanol in dichloromethane) indicated completion of the reaction and formation of a major product. The reaction mixture was quenched by diluting with DCM, followed by repetitive aqueous extractions to remove DMF and unreacted doxorubicin. The combined organic phase was dried over magnesium sulfate (MgSO 4 ), inorganic salts were filtered off and concentrated in vacuo to dryness. The crude material was purified by silica flash chromatography (20% v/v methanol in dichloromethane) to give the thiol reactive doxorubicin derivative, doxorubicin-maleimide, 47.7 mg (80% yield). The identity of the product was confirmed by high resolution LCMS and 1 H-NMR (supplementary information).

Protein expression and purification
His 6 -SUMO-LF N -DTA(C186S)-LPSTGG-His 5 , His 6 -SUMO-LF N -DTA(C186S), SrtA*-His 6 , wild-type protective antigen (PA), and PA[F427H] were expressed in E. coli BL21 (DE3) cells at New England Regional Center of Excellence/Biodefense and Emerging Infectious Diseases (NERCE). Each His 6 -tagged protein was purified using Ni-NTA columns. Each cell pellet (approximately 40 g) was resuspended in 100 ml of 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 buffer containing 200 mg lysozyme, 4 mg Roche DNAase I, and 2 tablets of Roche protease inhibitor cocktail. The suspension was sonicated on ice three times for 20 seconds. After sonication, the suspension was centrifuged at 17,000 rpm for 40 minutes. The lysate was loaded onto three 5 ml GE HisTrap FF crude Ni-NTA column pre-equilibrated with binding buffer (20 mM Tris-HCl pH 8.5, 150 mM NaCl, at pH 8.5). After loading the lysate, the columns were washed with 100 mL binding buffer then 100 mL 40 mM imidazole in 20 mM Tris-HCl pH 8.5, 500 mM NaCl. The protein was eluted using 500 mM imidazole in 20 mM Tris-HCl pH 8.5, 500 mM NaCl. The eluted protein was buffer exchanged to remove the imidazole using a HiPrep 26/10 Desalting column (GE Healthcare, UK). Wild-type PA and PA[F427H] were overexpressed in the periplasm of E. coli BL21 (DE3) cells and purified by anion exchange chromatography.

Sortase-mediated ligation
Sortase A was used to ligate peptides containing the N-terminal oligoglycine motif to LF N -DTA-LPSTGG (LDn). Staphylococcus aureus 59-206 SrtA (P94S/D160N/K196T; SrtA*) evolved by Chen, et al. was used for our sortase-mediated ligations. In order to obtain a native N-terminus, the small ubiquitin-like modified (SUMO) was cleaved off LFN-DTA-LPSTGG. SUMO cleavage was achieved using 1 µg SUMO protease per mg of protein substrate at RT for 1 hour followed by gel or LCMS analysis to confirm complete cleavage. We perform the sortasemediated ligations in the presence of Ni-NTA beads in order to bind all His 6 -tagged reagents and release the His 6 -free product in the supernatant. Ni-NTA beads were equilibrated with SrtA buffer (10 mM CaCl 2 , 50 mM Tris-HCl, 150 mM NaCl, pH 7.5). In one pot, 50 µM LF N -DTA-LPSTGG-His 5 , 5 µM SrtA*, and 300 µM G 5 -peptide were incubated with Ni-NTA beads in SrtA buffer for 30 min at RT while rotating. After incubation, the beads were spun down at 4 °C and the supernatant was collected. The beads were washed twice with SrtA buffer and twice with 10 mM imidazole in 20 mM Tris-HCl pH 7.5, 150 mM NaCl (to remove any non-specifically bound LF N ). The supernatant and all washes were combined, concentrated, and buffer exchanged three times into 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 to remove the excess G 5 -peptide. The purity of the ligated product (LDn) was analyzed by LCMS. Concentrations of the ligated products containing non-natural functionalities were determined using Bradford assay.

Sequence
Observed (