Sulaiman, Elias;
Yellon, Derek M;
Davidson, Sean M;
(2025)
A cautionary note on the potential pitfalls of using N-terminal truncated CD63 to label small extracellular vesicles.
Scientific Reports
, 15
, Article 7261. 10.1038/s41598-025-91597-6.
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Abstract
Small extracellular vesicles (sEV) are nanosized vesicles that facilitate intracellular communication. A significant research obstacle is the isolation of sEV devoid of non-sEV contaminants. Immunoaffinity capture with sEV-specific antibodies is an attractive approach to purifying sEV, but it risks disrupting the vesicles during antibody dissociation. Furthermore, immunoaffinity capture may require the modification of EV-specific proteins for the incorporation of tags on the EV surface, with unknown implications on EV production and function. The aim of this study was to investigate whether a previously reported CD63 truncation is efficient for the incorporation of small tags on the extravesicular surface. We therefore conjugated ALFA-tag to N-terminal-truncated CD63, and included nanoluciferase at the C-terminus, for luminescent tracing of the sEV. Full-length CD63-nanoluciferase was used as a control. Plasmid constructs expressing these proteins were transfected into HEK293 cells. In contrast to a previous report, the N-terminal truncation of CD63 impaired its membrane localisation and reduced the yield of EVs. Further investigation revealed that some of the tagged CD63 was co-localized with aggresomes and was preferentially secreted from the cells as soluble protein rather than being associated with sEV. These results demonstrate that CD63 truncation can impair its function and EV yield, potentially generating misleading results.
| Type: | Article |
|---|---|
| Title: | A cautionary note on the potential pitfalls of using N-terminal truncated CD63 to label small extracellular vesicles |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1038/s41598-025-91597-6 |
| Publisher version: | https://doi.org/10.1038/s41598-025-91597-6 |
| Language: | English |
| Additional information: | This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Cardiovascular Science UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Cardiovascular Science > Pre-clinical and Fundamental Science |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10207093 |
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