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Proteomics. 2019 Mar 13:e1700453. doi: 10.1002/pmic.201700453. [Epub ahead of print]

Surfaceome of exosomes secreted from the colorectal cancer cell line SW480: Peripheral and integral membrane proteins analyzed by proteolysis and TX114.

Author information

1
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Australia.
2
Department of Applied Biological Science, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan.
3
Global Innovation Research Organization, Tokyo University of Agriculture and Technology, Tokyo, Japan.

Abstract

Exosomes are bidirectional cell-cell communicators that play a crucial role in both normal and pathological physiology. Although exosomal surface membrane proteins (surfaceome) enable target cell recognition and are an attractive source of disease markers they are still poorly understood. Here, we describe a comprehensive surfaceome analysis of exosomes secreted by the colorectal cancer (CRC) cell line SW480 using a combination of sodium carbonate extraction / Triton X-114 phase separation approach as well as mild proteolysis of intact exosomes using proteinase K (PK). Label-free quantitative mass spectrometry revealed 1025 proteins of which 208 were predicted to be integral membrane proteins (IMPs) according to membrane protein topology and signal peptide prediction (TOPCONS) analysis and hydrophobicity distribution using Grand Average of hydropathy (GRAVY) scores. Interrogation of UniProt database-annotated proteins revealed 124 predicted peripherally-associated membrane proteins (PMPs). Surprisingly, 122 RNA-binding proteins (RBPs)/ RNA nucleoproteins (RNPs) were found in the carbonate/Triton X-114 insoluble fraction. Mild PK treatment of SW480-GFP labelled-exosomes revealed 58 proteolytically cleaved IMPs and 14 exoplasmic PMPs (e.g., CLU/ GANAB/LGALS3BP). A striking finding was 18 RBPs/RNPs (e.g., EIF3L/ RPL6) appear bound to the outer exosome surface since they were sensitive to PK proteolysis. Our finding that outer surface-localised 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. This article is protected by copyright. All rights reserved.

KEYWORDS:

exosomes; extracellular vesicles; integral membrane proteins; membrane shaving; surfaceome

PMID:
30865381
DOI:
10.1002/pmic.201700453

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