Purified horse milk exosomes contain an unpredictable small number of major proteins

Abstract

Exosomes are 40–100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel filtration. Individual preparations demonstrated different profiles of gel filtration showing well or bad separation of exosome peaks and one or two peaks of co-isolating proteins and their complexes. According to the electron microscopy, well purified exosomes displayed a typical exosome-like size (30–100 nm) and morphology. It was shown that exosomes may have several different biological functions, but detection of their biological functions may vary significantly depending on the presence of exosome contaminating proteins and proteins directly into exosomes. Exosome proteins were identified before and after gel filtration by MALDI MS and MS/MS spectrometry of protein tryptic hydrolyzates derived by SDS PAGE and 2D electrophoresis. The results of protein identification were unexpected: one or two peaks co-isolating proteins after gel-filtration mainly contained kappa-, beta-, alpha-S1-caseins and its precursors, but these proteins were not found in well-purified exosomes. Well-purified exosomes contained from five to eight different major proteins: CD81, CD63 receptors, beta-lactoglobulin and lactadherin were common to all preparations, while actin, butyrophilin, lactoferrin, and xanthine dehydrogenase were found only in some of them.The article describes the morphology and the protein content of major horse milk exosomes for the first time. Our results on the decrease of major protein number identified in exosomal preparations after gel filtration may be important to the studies of biological functions of pure exosomes.

Exosomes from human placenta purified by affinity chromatography on sepharose bearing immobilized antibodies against CD81 tetraspanin contain many peptides and small proteins

“…As mentioned above, exosome preparations obtained from different biological fluids using different types of centrifugation and ultracentrifugation can contain according to from a few dozen to several thousands of different proteins. However, as has been shown recently after additional purification of exosomes from coprecipitating proteins and their complexes by gel filtration, they contain no more than 10–12 major proteins ().…”

“…Our data shows that ferritin is not a part of exosomes, and should be considered as impurity even in preparations purified on anti‐CD81 Sepharose. Thus, there is a relatively small number of major proteins (Table ) in purified exosomes from human placenta, similar to exosomes from mare's milk .…”

“…We have recently shown that crude exosome preparations obtained from horse milk by several standard centrifugation and ultracentrifugation methods contain great number of proteins . After gel filtration, the peak corresponding to main part of impurity proteins, was separated from the peak of exosomes.…”

Extremely stable high molecular mass soluble multiprotein complex from eggs of sea urchin Strongylocentrotus intermedius with phosphatase activity

“…Stable multiprotein complex samples were applied for 1 minute for adsorption on a copper grid covered by formvar film stabilized with carbon and used for negative contrast according to Sedykh et al The grid was placed on a drop of 0.5% solution of uranyl acetate or 2% solution of phosphotungstic acid (5‐10 s); the excess of liquid was removed with filter paper. The grid was examined using a transmission electron microscope Jem1400 (Jeol, Japan), and images were obtained using a Veleta digital camera.…”

Small RNAs in eucaryotes: new clues for amplifying microRNA benefits

“…In the recent years, several studies revealed that human, bovine, pig and rat milk, as well as other biological fluids, contained miRNAs [82,83]. In particular, the miRNA microarray analyses of bovine milk revealed the presence of 79 and 91 miRNAs in the exosomes milk and in the supernatant whey derivatives, respectively [84]. Exosomes are naturally 30-100 nm nanovescicles containing different biomolecules including nucleic acids such as miRNAs and other ncRNAs, secreted into the extracellular liquids by different types of cells [85].…”