Stabilization of exosome-targeting peptides via engineered glycosylation

Abstract

Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics.

Keywords: Endosome; Exosome; Glycosylation; Peptides; Protease.

FIGURE 1.

Stability of exosome-targeting peptides. A, this graphic illustrates the orientation of Lamp2b in the endosomal membrane and exposure of N-terminal peptides (N.-term. peptide) to proteases. MVB, multivesicular body. C.-term., C terminus. B, transmission electron microscopy image of exosomes isolated by differential centrifugation from HEK293FT cell supernatant. C, size distribution of exosomes secreted by HEK293FT cells. D, enrichment of exosome-associated protein CD63 in exosome preparations relative to β-actin. E and F, expression of Lamp2b fusion proteins in cell lysates (E) and exosomes (F), as evaluated by HA (C-terminal) and FLAG (N-terminal) Western blots.

FIGURE 2.

Glycosylation motif-mediated stabilization of Lamp2b fusion proteins. A, expression of Lamp2b fusion proteins including an engineered GNSTM glycosylation motif in cells. In this and subsequent figures, the abbreviation “Xgs” is used to indicate a flexible linker X amino acids in length, comprising glycine and serine residues. B, transfection efficiency of cells expressing Lamp2b fusion proteins. Error bars indicate mean ± S.D. C and D, expression of Lamp2b fusion proteins in cell lysates (C) and exosomes (D) measured by HA (C-terminal) and FLAG (N-terminal) Western blots. E, cells were treated with either bafilomycin A1 (Baf.), which blocks endosomal acidification, or leupeptin, which inhibits endosomal proteases.

FIGURE 3.

Impact of engineered glycosylation motif on targeting peptide binding interactions. A and B, expression of FLAG-Lamp2b fusion proteins in cell lysates (A) and exosomes (B) before and after pulldown with anti-FLAG beads. C, lysates from cells expressing NST-tagged FLAG-Lamp2b proteins were diluted 1:5 in TBS (where indicated) and pulled down to confirm that apparent FLAG-mediated pulldown was not an artifact of variable levels of protein in the pulldown assay load. D, pulldown of intact exosomes requires that the FLAG tag be expressed on the Lamp2b N terminus (exosome exterior).

FIGURE 4.

Glycosylation-enhanced targeted delivery of exosomes to neuroblastoma cells. A, equivalent numbers of PHK67-labeled exosomes were incubated with Neuro2A cells for 2 h (∼3 × 10⁹ exosomes per 1 × 10⁵ cells), and uptake was quantified by flow cytometry. The shaded histogram is the exosome-free negative control to evaluate excess dye and dye-derived micelles. exos, exosomes. B, quantification of peaks presented in panel A.