Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

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.

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

“…The inability of cytotransmabs to internalize into HSPG-deficient cells demonstrated that their cellular uptake initiated via HSPGs as cell surface receptors, like the parent VLs, 16,17 though the precise HSPG molecule remains to be identified. HSPGs commonly serve as internalizing receptors for many macromolecules, including CPPs, anti-DNA antibodies, polycation-nucleic acid complexes, viruses, and growth factors.…”

“…16,17 The presence of exogenous heparin, which is a close structural homolog of HS, significantly inhibited the cellular internalization of TMab4 and HuT4 (Fig. 3B).…”

“…18 However, in live HeLa cells (human cervix carcinoma), hT0 failed to bind to cell surfaces, leading to complete loss of the cell-penetrating ability of m3D8 VL 16,17 (Fig. 1A).…”

“…15 The ability of m3D8 scFv to penetrate cells and then localize into the cytosol (referred to herein as 'cytosol-penetrating ability') was derived from the m3D8 VL. 16,17 Thus, m3D8 VL is an ideal moiety for the generation of cytosol-penetrating antibodies that can be used for diverse purposes.…”

Dual Targeting of the Chemokine Receptors CXCR4 and ACKR3 with Novel Engineered Chemokines

“…CXCR4 differs from CCR5 in that it is degraded through lysosomal pathways after agonist stimulation, whereas CCR5 is recycled back to the cell surface (59 -61). Because the fate of the internalized phage is largely dictated by the trafficking properties of the displayed protein (62), it is probable that phage particles displaying CXCL12 agonists were efficiently degraded and therefore not available for recovery. In cases where the receptor traffics through degradative pathways, using lysosomal inhibitors may permit selection of internalizing receptor agonists in future phage display experiments.…”