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 3β-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 micromolar) for one hour displayed ~ 8,400,000 NTA groups on the cell surface. Subsequent addition of the green fluorescent protein AcGFP fused to hexahistidine or decahistidine peptides (3 micromolar) and Ni(OAc) 2 (100 micromolar) enhanced the endocytosis of AcGFP by 150-fold (hexahistidine fusion protein) or 600-fold (decahistidine fusion protein) within four hours at 37 °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.Cell penetrating peptides and proteins (CPPs) have emerged as important new tools for the delivery of impermeable molecules into living mammalian cells. These delivery systems have been constructed from basic segments of HIV Tat, 1 oligoguanidine-containing peptoids, 2 β-peptides, 3 and other oligocationic motifs. 4 The mechanism of cellular penetration by CPPs is primarily thought to involve endocytosis, 5 particularly with larger cargo, 6 but varies depending on the delivery system and the cell type. 7Oligohistidine peptides are often fused to proteins to provide affinity tags ("His tags") that facilitate protein purification. To enable these peptides to function as CPPs, we synthesized an artificial cell surface receptor (1) comprising the plasma membrane anchor N-alkyl-3β-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). N-Alkyl-3β-cholesterylamine derivatives can function as prosthetic molecules active on the surface of living mammalian cells because this steroid can insert into cellular plasma membranes, project linked headgroups from the cell surface, and rapidly cycle between the plasma membrane and intracellular endosomes, similar to many naturally-occurring cell surface receptors. 8,9 The NTA motif binds tightly to nickel, cobalt, copper, and zinc dications, and immobilized metal chelate chromatography (IMAC) with NTA-linked supports is widely used to purify proteins fused to His tags. 10 Other lipids 11,12 and polymers 13 linked to NTA headgroups have also been reported. Receptor 1 was synthesized from 3β-cholesterylamine (2) 9 as shown in Scheme 1. Alkylation of 2 with ethyl 5-bromovalerate, followed by Boc protection to afford 3, provided an improved route to protected N-alkyl derivativ...

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Enhancing cell therapies from the outside in: Cell surface engineering using synthetic nanomaterials

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