Tutanov Oleg, Shtam Tatiana, Grigor'eva Alina, Tupikin Alexey, Tsentalovich Yuri, Tamkovich Svetlana
V. Zelman Institute for Medicine and Psychology, Novosibirsk State University, 630090 Novosibirsk, Russia.
Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Center "Kurchatov Institute", 188300 Gatchina, Russia.
Diagnostics (Basel). 2022 Mar 30;12(4):854. doi: 10.3390/diagnostics12040854.
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 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.
已知循环DNA（cirDNA）通过与DNA形成大分子复合物的蛋白质来免受核酸酶活性的影响。此外，先前的研究表明cirDNA可以结合到外泌体的外表面。NTA分析和实时PCR显示，来自健康女性（HF）或乳腺癌患者（BCP）血浆的外泌体含有少于1.4×10 pg的DNA。因此，只有一小部分cirDNA作为囊泡冠的一部分附着在外泌体的外侧：外泌体DNA的比例不超过HF血浆DNA的0.025%和BCP血浆DNA的0.004%。用DNase I处理血浆外泌体并随后进行斑点免疫测定表明，H2a、H2b和H3组蛋白不是外泌体膜的一部分，而是通过与DNA结合蛋白或组蛋白结合蛋白相互作用与外泌体表面相关的cirDNA-蛋白质大分子复合物的一部分。通过MALDI-TOF质谱鉴定后使用生物信息学方法，鉴定出16种外泌体DNA结合蛋白。结果表明，四种蛋白——AIFM1、IGHM、CHD5和KCNIP3——是外泌体外膜上DNA结合的候选蛋白；外泌体的冠可能包括五种DNA结合蛋白：H2a、H2b、H3、IGHM和ALB。值得注意的是，AIFM1、IGHM和CHD5蛋白仅在HF血浆外泌体中发现；KCNIP3蛋白仅在BCP血浆外泌体中鉴定到；而H2a、H2b、H3和ALB在所有血浆外泌体样本中均有发现。在HF血浆的外泌体中发现了两种组蛋白结合蛋白，CHD5和KDM6B。获得的数据表明，cirDNA通过与DNA结合蛋白结合优先结合到外泌体的外膜上。
