Abstract
Exosomes are nanovesicles (30–100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30–100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins’ tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11–13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes.
Highlights
Human placenta is a very highly specialized organ that connects mother and fetus organisms, and placental dysfunction could lead to catastrophic consequences for both
Eight major proteins were identified using MALDI mass spectrometry and MS/MS of tryptic protein hydrolysates corresponding the protein bands after one-dimensional SDS-PAGE of the proteins of first peak of exo-1 separated by the gel-filtration: hemoglobin, ferritin, CD63, CD81, alkaline phosphatase, human serum albumin (HSA), alpha-actin-4 and IgGs (Figure 5B, −DTT, peak 1, Table 1)
Alpha-actinin-4 IgG H and L chains c a MS—determination of a set of peptides from tryptic hydrolysates by MS/MS in accordance with the sequences of peptides. b For the identification of proteins and their molecular masses, the 2016 SwissProt program was used. c Unlike other proteins, ferritin, HSA, and IgGs are absent in exosome preparations after their trypsin treatment; apparently, these proteins are not internal exosome proteins
Summary
Human placenta is a very highly specialized organ that connects mother and fetus organisms, and placental dysfunction could lead to catastrophic consequences for both. An even more improbable result was obtained when analyzing proteins of the milk’s exosomes of cows, whose preparations were isolated by centrifugation and ultracentrifugation in a sucrose gradient [35]. We recently showed that human milk and placenta contain very stable high molecular weight (~1000 kDa) multiprotein complexes comparable in size to exosomes [37,38]. After additional purification of exosomes from stable high-molecular complexes and other proteins using gel filtration, only 7–8 different major proteins were detected in the composition of the milk, depending on milk sample [40]. Exosome preparations without additional purification may contain impurities of a very different nature, including proteins and nucleic acids. We analyzed the exosome proteins obtained from four human placentas using standard approaches of centrifugation and purified by gel filtration. We obtained unexpected results: exosomes isolated from placentas contain only 10–12 major proteins
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