Abstract
Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.
Highlights
Eukaryotic endocytosis of exogenous proteins, toxins, bacteria, and viral particles occurs through a vesicular transport mechanism facilitating transfer of the polar macromolecules across non-polar, lipophilic cell membranes
The recombinant phage displays 2–4 copies of full-length pIII derived from the helper phage and usually 1–2 copies of the recombinant pIII fusion proteins 3D8 variable single domain (VL)-pIII, TAT-pIII, or hAY4 scFv-pIII derived from the phagemid; the relative display ratio depends on the passenger proteins and culture conditions [33]
To determine the relative display level of fusion proteins, an equivalent titer of recombinant phages (1010 or 109 colony-forming units (CFU)) was analyzed by Western blotting using anti-myc antibody to detect only the pIII-fusion proteins or anti-pIII antibody to detect both pIII-fusion and intact full-length pIII proteins. 3D8 VL-pIII and hAY4 scFv-pIII were readily detected at a titer as low as 109 CFU, whereas TAT-pIII was weakly detected at 1010 CFU, but was negligible at 109 CFU (Fig. 1B)
Summary
Eukaryotic endocytosis of exogenous proteins, toxins, bacteria, and viral particles occurs through a vesicular transport mechanism facilitating transfer of the polar macromolecules across non-polar, lipophilic cell membranes. Cell-penetrating peptides (CPPs), such as the TAT peptide derived from HIV-1 transactivator protein, have the ability to penetrate into living mammalian cells [3,4] Because of their cationic nature, cationic CPPs, including TAT, utilize negatively charged glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) as cell surface receptors for the cellular uptake [5]. GAGs are covalently linked to cell surface core proteins such as syndecan and glypican forming HS proteoglycan (HSPG) and CS proteoglycan (CSPG) [6]. These anionic proteoglycans due to abundant carboxyl and sulfate groups in HS and CS are present on the surface of virtually all mammalian cells, explaining the cell type-independent internalizing ability of cationic CPPs via electrostatic interactions [3]. Despite consensus on sulfated proteoglycans as the cell surface receptor, the endocytosis of the TAT peptide remains controversial [4] and has been proposed by several mechanisms, such as clathrinmediated endocytosis [7], caveolae-mediated endocytosis [8], or macropinocytosis [5]
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