Transferrin receptor expression in serum exosomes as a marker of regenerative anaemia in the horse

Rout, Emily D.; Webb, Teckla L.; Laurence, H. M.; Long, Lindsey J.; Olver, Christine S.

doi:10.1111/evj.12235

Cited by 15 publications

(19 citation statements)

References 24 publications

(27 reference statements)

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“…Our data are in a good agreement with the previously published results of the analysis of exosomes isolated using successive centrifugation according to the adopted schemes from various biological fluids ; exosome preparations obtained in such way, were used in most of the published studies . Analysis of more than 200 vesicle preparations obtained from various biological fluids in addition to exosomes revealed in all preparations contaminating structures of low electron density that do not have membrane envelope and, therefore respectively, are not exosomes (“nonvesicles”) .…”

Section: Resultssupporting

confidence: 90%

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

et al. 2018

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Exosomes are nanovesicles (40-100 nm) containing various RNAs and different proteins. Exosomes are involved in intracellular communication and immune system function. Exosomes from different sources are usually isolated using standard methods-centrifugation and ultracentrifugations. Exosomes isolated by these procedures were reported to contain from a few dozen to thousands of different proteins. Here crude vesicle preparations from five placentas (normal pregnancy) were first obtained using standard centrifugation procedures. According to electron-microscopic studies, these preparations contained vesicles of different size (30-225 nm), particles of round shape of average electron density ("nonvesicles" 20-40 nm) (A), structured clusters of associated proteins and shapeless aggregations (B), as well as ring-shaped 10-14 nm structures formed by ferritin (C). After additional purification of the vesicle preparations by gel filtration on Sepharose 4B, the main part of protein structures was removed; however, the preparations still contained small admixtures of components A-C. Further purification of the preparations by affinity chromatography on Sepharose bearing immobilized antibodies against exosome surface protein CD81 led to isolation of highly purified exosomes (40-100 nm). These exosomes according to electron microscopy data contained tetraspanin embedded in the membrane, which was stained with antibodies against CD81 conjugated with 10-12 nm gold nanoparticles. SDS-PAGE and MALDI MS and MS/MS mass spectrometry of tryptic hydrolysates of proteins contained in these exosomes revealed eleven major proteins (>10 kDa):hemoglobin subunits, CD81, interleukin-1 receptor, annexin A5, cytoplasmic actin, alpha-actin-4, alkaline phosphatase, human serum albumin, serotransferrin, and lactotrasferrin. Using MALDI mass analysis of the highly purified exosomes, we for the first time found that in addition to the large proteins (>10 kDa), exosomes having affinity to CD81 contain more than 27 different peptides and small proteins of 2-10 kDa.This finding can be useful for revealing biological functions of pure exosomes. © 2018 IUBMB Life, 70(11):1144Life, 70(11): -1155Life, 70(11): , 2018 Additional Supporting Information may be found in the online version of this article.

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Section: Resultssupporting

confidence: 90%

“…The most efficient procedures include centrifugation with following ultracentrifugation, ultracentrifugation with density gradients etc. . Protocols for purification of exosomes by ultracentrifugation in a 30% sucrose/deuterium oxide cushion and after incubation in the ice at acidic pH has been published .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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“…To date, no data concerning horse milk exosomes have been published. At the same time, the only article devoted to horse exosomes evaluates these vesicles as markers of erythrocyte regeneration, as horses do not release reticulocytes into the peripheral blood [23] . The authors have proposed that transferrin receptor 1 (TfR1) expressed in exosomes of serum can provide a regeneration of new marker for anemic horses.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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“…Currently, extensive studies have been undertaken to elucidate the role of specific miRNAs in diverse disease processes and their potential use as biomarkers (53-58). Recently, equine miRNAs in normal tissues have been investigated (59,60), and exosomes and exosomal miRNAs derived from serum and ovarian follicular fluid in mares have been characterized (61)(62)(63). Even though SEs have been described for the horse and their role during sperm capacitation has been studied (46,64), their RNA cargo and function remain to be characterized.…”

mentioning

confidence: 99%

Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus

Carossino

Dini

Kalbfleisch

et al. 2018

J Virol

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Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the gene () and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented here suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion. Equine arteritis virus (EAV) has the ability to establish long-term persistent infection in the stallion reproductive tract and to be shed in semen, which jeopardizes its worldwide control. Currently, the molecular mechanisms of viral persistence are being unraveled, and these are essential for the development of effective therapeutics to eliminate persistent infection. Recently, it has been determined that long-term persistence is associated with a specific allele of the gene () and is maintained despite induction of local inflammatory, humoral, and mucosal antibody responses. This study demonstrated that long-term persistence is associated with the downregulation of seminal exosome miRNA eca-mir-128 and enhanced expression of its putative target, CXCL16, in the reproductive tract. For the first time, this study suggests complex interactions between eca-mir-128 and cellular elements at the site of EAV persistence and implicates this miRNA in the regulation of the CXCL16/CXCR6 axis in the reproductive tract during long-term persistence.

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Transferrin receptor expression in serum exosomes as a marker of regenerative anaemia in the horse

Abstract: These data indicate that TfR1 expression in serum exosomes may provide a marker for regeneration in anaemic horses.

Cited by 15 publications

References 24 publications

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

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

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

Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus

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