Year: 2019
Type: article
Abstract: Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum "extra-depletion" protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood.
Institutions: Johns Hopkins University, Johns Hopkins Medicine, Inserm, Université Paris Sciences et Lettres, Institut Curie, Centre National de la Recherche Scientifique, Génétique et biologie du développement, Universidad de Buenos Aires, Instituto de Investigaciones Biomédicas en Retrovirus y Sida
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $4,410
Funders: Sidaction, Agence Nationale de la Recherche, Agence Nationale de Recherches sur le Sida et les Hépatites Virales, National Institutes of Health, National Institute on Aging, National Institute of Mental Health, National Institute on Drug Abuse, National Institute of Allergy and Infectious Diseases
Awards: G1207648242, G2227547912, G2305452004, G2344771908, Paris Sciences et Lettres, Extracellular Vesicle-Mediated Immunomodulation in HIV Infection and Nicotine Abuse, Novel Approaches to Capture, Sorting, and Characterization of CNS-Origin Extracellular Vesicles, Biologie des cellules dendritiques, G6079011624, G6759105742, Discovering novel players in mechanisms of extracellular vesicle release, cargo loading, and early pathogenesis of late-onset Alzheimer's Disease, Next-generation extracellular vesicle biologics to target central nervous system and peripheral reservoirs of HIV, G8773427133, Extracellular Vesicle and Extracellular RNA Biomarkers of HIV-1 Central Nervous System Pathogenesis and Cigarette Use, Training Veterinarians for Careers in Biomedical Research, Novel Approaches to Capture, Sorting, and Characterization of CNS-Origin Extracellular Vesicles
OpenAlex ID: W2955442076