title: 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.
authors: From the Interdisciplinary Biological Sciences Program.
authors: Michelle E
authors: ME
authors: Hung
authors: Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, and Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, Illinois 60208 j-leonard@northwestern.edu.
authors: Joshua N
authors: JN
authors: Leonard
mesh_terms: D011992:Endosomes / Q000378:metabolism; D055354:Exosomes / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D052119:Lysosomal-Associated Membrane Protein 2 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D021381:Protein Transport; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: C497811:LAMP2 protein, human; D052119:Lysosomal-Associated Membrane Protein 2; D010455:Peptides; D011993:Recombinant Fusion Proteins
keywords: Endosome; Exosome; Glycosylation; Peptides; Protease
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