Surfaceome of Exosomes Secreted from the Colorectal Cancer Cell Line SW480: Peripheral and Integral Membrane Proteins Analyzed by Proteolysis and TX114

Abstract

Exosomes are important bidirectional cell–cell communicators in normal and pathological physiology. Although exosomal surface membrane proteins (surfaceome) enable target cell recognition and are an attractive source of disease marker, they are poorly understood. Here, a comprehensive surfaceome analysis of exosomes secreted by the colorectal cancer cell line SW480 is described. Sodium carbonate extraction/Triton X‐114 phase separation and mild proteolysis (proteinase K, PK) of intact exosomes is used in combination with label‐free quantitative mass spectrometry to identify 1025 exosomal proteins of which 208 are predicted to be integral membrane proteins (IMPs) according to TOPCONS and GRAVY scores. Interrogation of UniProt database‐annotated proteins reveals 124 predicted peripherally‐associated membrane proteins (PMPs). Surprisingly, 108 RNA‐binding proteins (RBPs)/RNA nucleoproteins (RNPs) are found in the carbonate/Triton X‐114 insoluble fraction. Mild PK treatment of SW480‐GFP labeled exosomes reveal 58 proteolytically cleaved IMPs and 14 exoplasmic PMPs (e.g., CLU/GANAB/LGALS3BP). Interestingly, 18 RBPs/RNPs (e.g., EIF3L/RPL6) appear bound to the outer exosome surface since they are sensitive to PK proteolysis. The finding that outer surface‐localized miRNA Let‐7a‐5p is RNase A–resistant, but degraded by a combination of RNase A/PK treatment suggests exosomal miRNA species also reside on the outer surface of exosomes bound to RBPs/RNPs.

The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles

“…A role in the modulation of autophagy was also revealed for the heterogeneous nuclear ribonucleoprotein K (HNRNPK) and M (HNRNPM) [61,64]. Further ribosomal proteins-namely RPL5, RPL6, RPL12, RPL18, RPL22, RPS16, and RPS4X-were identified, possibly due to their RNA-binding property or, as recently reported, being present on the surface of exosomes [65].…”

Sustained subcutaneous delivery of secretome of human cardiac stem cells promotes cardiac repair following myocardial infarction

“…29 EVs may act as signaling complexes by direct stimulation of target cells, by transferring surface receptors between cells, and by delivering proteins, mRNA and bioactive lipids into the target cells. Recently, the surfaceome of EVs has been characterized, which has provided insights into the tropism capacity of EVs, 30 such as through various exosomal integrins to mediate organ-specific delivery. 31 We have utilised global cell surface expression and cardiac subcellular membrane studies to correlate key proteins associated within EVs as potential molecular bias for cardiac tropism and cell surface interaction.…”

Targeting Vesicular LGALS3BP by an Antibody-Drug Conjugate as Novel Therapeutic Strategy for Neuroblastoma

“…Recent studies have revealed that LGALS3BP plays a key role in the cross-talk between neuroblastoma cells and the tumor microenvironment (TME) [ 17 ]. Indeed, LGALS3BP is one of the most abundant surface component of cancer-derived EVs [ 18 , 19 , 20 ]. EVs have a prominent role in inducing a TME that is favorable to cancer progression through the induction of immune escape, stimulation of angiogenesis and preparation of the pre-metastatic niche [ 21 , 22 ].…”