Synthesis of an artificial cell surface receptor that enables oligohistidine affinity tags to function as metal-dependent cell-penetrating peptides

Abstract

Cell-penetrating peptides and proteins (CPPs) are important tools for the delivery of impermeable molecules into living mammalian cells. To enable these cells to internalize proteins fused to common oligohistidine affinity tags, we synthesized an artificial cell surface receptor comprising an N-alkyl derivative of 3beta-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). This synthetic receptor inserts into cellular plasma membranes, projects NTA headgroups from the cell surface, and rapidly cycles between the plasma membrane and intracellular endosomes. Jurkat lymphocytes treated with the synthetic receptor (10 microM) for 1 h displayed approximately 8,400,000 [corrected]NTA groups on the cell surface. Subsequent addition of the green fluorescent protein AcGFP fused to hexahistidine or decahistidine peptides (3 microM) and Ni(OAc)(2) (100 microM) enhanced the endocytosis of AcGFP by 150-fold (hexahistidine fusion protein) or 600-fold (decahistidine fusion protein) within 4 h at 37 degrees C. No adverse effects on cellular proliferation or morphology were observed under these conditions. By enabling common oligohistidine affinity tags to function as cell-penetrating peptides, this metal-chelating cell surface receptor provides a useful tool for studies of cellular biology [corrected]

Figure 1

Top: Structure of the synthetic cell surface receptor (1) that binds metals and His tags. Bottom: Strategy for delivery of His-tagged proteins into mammalian cells by synthetic receptor-mediated endocytosis.

Figure 2

Confocal laser scanning (left) and differential interference contrast (right) micrographs of living Jurkat lymphocytes. Cellular plasma membranes were preloaded with receptor 1 (10 μM) for 1 h at 37 °C, cells were washed with fresh media, and media containing AcGFP-His₁₀ (3.2 μM) and Ni(OAc)₂ (100 μM) was added for an additional 4 h at 37 °C. Cells shown in Panel A were imaged immediately after this treatment. Cells shown in Panel B were washed with NTA (400 μM, 30 min) prior to microscopy to remove non-internalized protein from the cell surface. Scale bar = 10 μm.

Figure 3

Analysis of cellular uptake of His-tagged AcGFP proteins by flow cytometry. Each bar represents the average fluorescence of 10,000 cells. Unless otherwise noted, cellular plasma membranes of Jurkat lymphocytes were preloaded with receptor 1 (10 μM) for 1 h at 37 °C, cells were washed with fresh media, and His-tagged AcGFP (3.2 μM)/metal diacetate solution (100 μM) was added for 4 h at 37 °C. Prior to analysis, cells were washed with NTA (400 μM, 30 min) in PBS (pH 7.4) to remove bound surface protein. Panel A: Dose dependence of receptor-mediated uptake. Premix conditions: a solution containing receptor 1, Ni(OAc)₂, and AcGFP(His)₁₀ was added to cells for 4 h. Panel B: Dependence on [Ni(OAc)₂]. Panel C: Omission control experiments. Panel D; Uptake of (His)₆ and (His)₁₀ fusion proteins promoted by different metal diacetates.

Scheme 1

Reagents and conditions: (a) ethyl 5-bromovalerate, K₂CO₃, DMF, 60 °C; (b) (Boc)₂O, DIEA, CH₂Cl₂; (c) N-Fmoc-β-Ala, EDC, HOBt, DIEA, CH₂Cl₂; (d) piperidine, DMF; (e) LiOH·H₂O, MeOH/THF/H₂O (3:2:1); (f) 6, EDC, HOBt, CH₂Cl₂; (g) N^α, N^α-bis[(tert-butoxycarbonyl)methyl]-L-Lys tert-butyl ester, EDC, HOBt, CH₂Cl₂; (h) TFA, CH₂Cl₂.