Blood Plasma Exosomes Contain Circulating DNA in Their Crown

Abstract

It is known that circulating DNA (cirDNA) is protected from nuclease activity by proteins that form macromolecular complexes with DNA. In addition, it was previously shown that cirDNA can bind to the outer surface of exosomes. NTA analysis and real-time PCR show that exosomes from healthy females (HF) or breast cancer patients (BCP) plasma contain less than 1.4 × 10−8 pg of DNA. Thus, only a minor part of cirDNA is attached to the outer side of the exosome as part of the vesicle crown: the share of exosomal DNA does not exceed 0.025% HF plasma DNA and 0.004% BCP plasma DNA. Treatment of plasma exosomes with DNase I with subsequent dot immunoassay reveals that H2a, H2b, and H3 histones are not part of the exosomal membrane, but are part of the cirDNA–protein macromolecular complex associated with the surface of the exosome either through interaction with DNA-binding proteins or with histone-binding proteins. Using bioinformatics approaches after identification by MALDI-TOF mass spectrometry, 16 exosomal DNA-binding proteins were identified. It was shown that four proteins—AIFM1, IGHM, CHD5, and KCNIP3—are candidates for DNA binding on the outer membrane of exosomes; the crown of exosomes may include five DNA-binding proteins: H2a, H2b, H3, IGHM, and ALB. Of note, AIFM1, IGHM, and CHD5 proteins are found only in HF plasma exosomes; KCNIP3 protein is identified only in BCP plasma exosomes; and H2a, H2b, H3, and ALB are revealed in all samples of plasma exosomes. Two histone-binding proteins, CHD5 and KDM6B, have been found in exosomes from HF plasma. The data obtained indicate that cirDNA preferentially binds to the outer membrane of exosomes by association with DNA-binding proteins.

The Influence of Proteins on Fate and Biological Role of Circulating DNA

“…It was found that cirDNA binds to the outer membrane of exosomes by association with DNA-binding proteins. However, the share of exoDNA does not exceed 0.025% and 0.004% in healthy female and breast cancer patients' plasma DNA, respectively [30].…”

“…While the presence of DNA in the EV lumen is currently a very debatable question in the scientific community, there is increasing evidence of cirDNA and cirRNA present on the outer surface of small EVs (including exosomes-vesicles with a size of 30-150 nm, carrying tetraspanins such as CD9, CD63, and CD81 on their membrane) bound by nucleic acid binding proteins. In our previous work, we have shown the presence of DNA on the outer membrane of exosomes, and have identified several membrane DNA-binding and histone-binding proteins in small EV proteomes (AIFM1, IGHM, CHD5, KCNIP3, CHD5, and KDM6b, respectively) [30]. However, it was shown that the share of that cirDNA fraction did not exceed 0.025% in the total pool of cirDNA from the blood of healthy females and breast cancer patients, suggesting that this type of circulation barely contributes to cirDNA circulation in general.…”

“…2022, 23, x FOR PEER REVIEW 3 of 16 cleic acid binding proteins.. In our previous work, we have shown the presence of DNA on the outer membrane of exosomes, and have identified several membrane DNA-binding and histone-binding proteins in small EV proteomes (AIFM1, IGHM, CHD5, KCNIP3, CHD5, and KDM6b, respectively) [30]. However, it was shown that the share of that cirDNA fraction did not exceed 0.025% in the total pool of cirDNA from the blood of healthy females and breast cancer patients, suggesting that this type of circulation barely contributes to cirDNA circulation in general.…”

“…Several structures involved with cirDNA circulation in biological fluids have been described to date: EVs (small EVs or exosomes, large EVs or microvesicles, apoptotic bodies, etc.) [30], macromolecular complexes (complexes with nucleosomes, other lipids, and proteins, e.g., serum proteins) [32,33], and blood cell surface bound cirDNA [34] (Figure 2). Such association of cirDNA might allow it to be better protected from blood endonucleases, thus allowing for the increase of its half-life in circulation, while the addition of purified deoxyribo-oligonucleotides and DNA amplicons to blood plasma leads to their rapid elimination due to endogenous blood nucleases [35].…”

Exosomes in Breast Cancer: Involvement in Tumor Dissemination and Prospects for Liquid Biopsy

“…However, many researchers suppose that the presence of DNA inside exosomes is an artifact related to the contamination of vesicle preparations by apoptotic blebs of similar size. Recent works demonstrate the presence of DNA on the exosomal outlier membrane surface, which binds to the vesicle membrane through DNA-binding proteins [ 64 , 65 ]. Exosomes of infected cells also often carry pathogen DNA, inducing an adaptive immune response in lymphocytes by triggering the cGAS-STING signaling pathway and similarly triggering the anti-tumor immune response [ 66 , 67 ].…”