Efficient Delivery of Streptavidin to Mammalian Cells: Clathrin-Mediated Endocytosis Regulated by a Synthetic Ligand
journal contributionposted on 04.05.2002, 00:00 by Stephen L. Hussey, Blake R. Peterson
The efficient delivery of macromolecules to living cells presents a formidable challenge to the development of effective macromolecular therapeutics and cellular probes. We describe herein a novel synthetic ligand termed “Streptaphage” that enables efficient cellular uptake of the bacterial protein streptavidin by promoting noncovalent interactions with cholesterol and sphingolipid-rich lipid raft subdomains of cellular plasma membranes. The Streptaphage ligand comprises an N-alkyl derivative of 3β-cholesterylamine linked to the carboxylate of biotin through an 11-atom tether. Molecular recognition between streptavidin and this membrane-bound ligand promotes clathrin-mediated endocytosis, which renders streptavidin partially intracellular within 10 min and completely internalized within 4 h of protein addition. Analysis of protein uptake in Jurkat lymphocytes by epifluorescence microscopy and flow cytometry revealed intracellular fluorescence enhancements of over 300-fold (10 μM ligand) with >99% efficiency and low toxicity. Other mammalian cell lines including THP-1 macrophages, MCF-7 breast cancer cells, and CHO cells were similarly affected. Structurally related ligands bearing a shorter linker or substituting the protonated steroidal amine with an isosteric amide were ineffective molecular transporters. Confocal fluorescence microscopy revealed that Streptaphage-induced uptake of streptavidin functionally mimics the initial cellular penetration steps of Cholera toxin, which undergoes clathrin-mediated endocytosis upon binding to the lipid raft-associated natural product ganglioside GM1. The synthetic ligand described herein represents a designed cell surface receptor capable of targeting streptavidin conjugates into diverse mammalian cells by hijacking the molecular machinery used to organize cellular membranes. This technology has potential applications in DNA delivery, tumor therapy, and stimulation of immune responses.
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noncovalent interactionsTHP4 hStreptaphage ligand10 minflow cytometrycell linesproduct ganglioside GM 1.protein additionConfocal fluorescence microscopyprotein streptavidinplasma membranesDNA deliveryprotein uptakeCholera toxinSynthetic Ligandepifluorescence microscopyprotonated steroidal amineisosteric amidetumor therapyCHO cellsstreptavidin conjugatesMolecular recognitioncell surface receptorJurkat lymphocytesEfficient DeliveryMCFpenetration stepsintracellular fluorescence enhancements