Isolation and characterization of extracellular vesicles: proteomic insights

Thesis with publications.

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science (LIMS), School of Molecular Sciences, Faculty of Science, Health and Engineering, La Trobe University, Bundoora.

Secretion and exchange of extracellular vesicles (EVs) is a central paradigm for intercellular communication. EVs comprise at least two major subtypes: exosomes (Exos) and shed microvesicles (sMVs). Both contain protein, DNA, RNA and lipid cargoes, which upon uptake by recipient cells play a critical physiological role in healthy and physiopathological conditions. In this thesis I tackled several key problems hindering EV research. The first involved developing a facile procedure for separating and characterizing individual EV subtypes, a necessary prerequisite for determining their biological function. In Chapter 2 I developed a sequential centrifugal ultrafiltration procedure for separating Exos and sMVs using the colorectal cancer (CRC) cell line LIM1863 as a model. Label-free quantitative MS-based proteomics identified 350 proteins uniquely enriched in sMVs compared to exosomes. Importantly, many of these distinguish sMVs from exosomes (e.g., KIF23, CSE1L, and RACGAP1). This is the first report of stereotypic marker proteins for sMVs. Next, I performed a bioinformatic analysis of RNA binding proteins (RBPs) and ribonucleoprotein (RNP) complexes in LIM1863-derived EV subtypes using previously published proteomic and deep sequencing data (Chapter 3). This analysis is a first step towards shedding light on the possible biological role of RNP complexes in EVs. In chapter 4, I performed a comprehensive proteome analysis of Exos to investigate the spatial organization of proteins and RNAs in exosomes; the first step towards developing an Exosome Proteome database. Na2CO3/Triton X-114 detergent phase separation (DPS) was used to enrich for membrane-associated SW480-derived exosomal proteins and proteinase K ‘shaving’ (PKS) to discriminate exosomal surface proteins from luminal proteins. DPS of SW480-derived exosomes identified 805 proteins, of which 179 were classified as membrane-associated proteins based on TMHMM/GRAVY analysis; 73 novel membrane proteins (e.g., TSPAN6, ADAM15, MMP14) previously not seen in XII SW480-Exos were identified. A salient finding was that Exos-surface-bound RBPs (DDX17, hnRNPA1, DDX39B, DHX9) are readily shaved by proteinase K thereby exposing Exos-miRs to RNase A degradation; importantly, luminal RBPs involved in mRNA binding are resistant to RNase treatment. In Chapter 5 I focused on the claudin (CLDN) family of membrane associated tetraspanins, which are key components of the tight junction paracellular permeability barrier of polarised epithelial cells and are also implicated in CRC progression. Using the human isogenic CRC cell lines SW480 (primary carcinoma) and metastatic SW620 I showed that the claudin family members disproportionately distribute to SW480/SW620 exosomes. Using anti-CLDN7-pAb and anti-CLDN1-mAb I performed Co-IP-MS analysis on SW480-Exos and SW620-Exos, respectively. A key finding was the identification of an exosomal CLDN assembly complex CLDN-7/ EPCAM/ CD44 - a cellular complex known to promote CRC progression. Collectively, findings from my thesis provide novel insights into the biophysical/ biochemical properties of exosomes and sMVs.