Detailed Analysis of Protein Topology of Extracellular Vesicles-Evidence of Unconventional Membrane Protein Orientation

Abstract

Extracellular vesicles (EVs) are important mediators of intercellular communication that change the recipient cell by shuttling lipids, RNA, or protein cargo between cells. Here, we investigate the topology of the protein cargo found in EVs, as this topology can fundamentally influence the biological effects of EVs. A multiple proteomics approach, combining proteinase treatment and biotin tagging, shows that many proteins of cytosolic origin are localized on the surface of EVs. A detailed analysis of the EV proteome at the peptide level revealed that a number of EV membrane proteins are present in a topologically reversed orientation compared to what is annotated. Two examples of such proteins, SCAMP3 and STX4, were confirmed to have a reversed topology. This reversed typology was determined using flow cytometry and fluorescent microscopy with antibodies directed toward their cytoplasmic epitopes. These results describe a novel workflow to define the EV proteome and the orientation of each protein, including membrane protein topology. These data are fundamentally important to understanding the EV proteome and required to fully explain EV biogenesis as well as biological function in recipient cells.

Figure 1. Schematic illustration of study.

EVs from HMC-1 cells were treated with proteinase K (PK) or trypsin/Lys-C. Trypsin/Lys-C treated EVs were further treated with sulfo-LC-biotin, and biotinylated peptides were isolated by column based separation. EVs, PK-treated EVs, and biotinylated peptides were analyzed with LC-MS/MS.

Figure 2. Characterization of EVs and PK-treated EVs.

(a) Western blot analysis with EV markers, CD81 and TSG101, in 12 fractions from OptiPrep density gradient. (b) The particle number in each OptiPrep fraction was analyzed by nanoparticle tracking analysis. (c) Cryo-EM images of non-treated and PK-treated EVs. (d) Western blot analysis of non-treated and PK-treated EVs with CD81 and beta-actin.

Figure 3. Defining EV and surface-accessible proteome.

(a) Venn diagram of non-treated and PK-treated EVs proteome. The number present in the circle represent the total number of identified proteins in particular data set. (b) Plot of log2 value of relative abundance of proteins from non-treated and PK-treated EVs. Line and dotted line indicate half and 2-fold change, respectively. (c) Among the common proteins, proteins are divided into three groups–2-fold increase, 2-fold decrease, and no change after PK treatment–based on relative protein abundance. (d) Hierarchical diagram of defining EV and surface-accessible proteome.

Figure 4. Subcellular localization and gene ontology analysis of EV and surface-accessible proteome.

(a) Subcellular localization of EV and surface-accessible proteome. (b) Top 10 biological process gene ontology terms enriched in EV proteome. (c) Top 10 biological process gene ontology terms enriched in surface-accessible proteome.

Figure 5. Topology analysis of transmembrane and lipid-anchored proteins.

(a) Hierarchical diagram of defining topology of transmembrane and lipid-anchored proteins. Proteins were visualized with Protter tool and the information about the identified peptides was integrated. Based on localization of peptides, correct topology and inside-out were defined. (b) Topology illustration of two examples of conclusively inside-out proteins. Peptides that were found in LC-MS/MS were visualized by Protter.

Figure 6. Validation of surface-accessible proteome and inside-out proteins.

(a) Western blot analysis of surface-accessible and EV proteome. STUB1, GAPDH, Histone H1, and PCNA are surface-accessible proteome, whereas Flotilin 1 and TSG101 are EV proteome. Relative band intensity was measured. (b) Flow cytometry of inside-out membrane proteins. EVs were captured by SCAMP3 (left panel) or STX4 (right panel) antibody conjugated beads and then detected with CD63 or CD81. (c) The percentage of SCAMP3, STX4, and beta-actin positive EVs were calculated after incubation with or without 0.1% Tween-20.

Figure 7. Schematic overview of EV proteome.

Selected examples of proteins are illustrated. Proteins in EV isolates can be localized either inside (blue box) or on the surface (gray box) of EVs. In addition, some of transmembrane and lipid-anchored proteins have inside-out topology (green box).