Exosomes: from biogenesis and secretion to biological function.
ABSTRACT Exosomes are small microvesicles that are released from late endosomal compartments of cultured cells. Recent work has shown that exosome-like vesicles are also found in many body fluids such as blood, urine, ascites and amnionic fluid. Although the biological function of exosomes is far from being fully understood, exosomes may have general importance in cell biology and immunology. The present review aims to address some of the facets of exosome research with particular emphasis on the immunologist's perspective: (i) exosomes as a novel platform for the ectodomain shedding of membrane proteins by ADAMs and (ii) recent findings on the role of exosomes in tumor biology and immune regulation.
- SourceAvailable from: Haroldo Oliveira[Show abstract] [Hide abstract]
ABSTRACT: Paracoccidioides species are dimorphic fungi and are the etiologic agents of paracoccidioidomycosis, which is a serious disease that involves multiple organs. The many tissues colonized by this fungus suggest a variety of surface molecules involved in adhesion. A surprising finding is that most enzymes in the glycolytic pathway, tricarboxylic acid (TCA) cycle and glyoxylate cycle in Paracoccidioides spp. have adhesive properties that aid in interacting with the host extracellular matrix and thus act as 'moonlighting' proteins. Moonlighting proteins have multiple functions, which adds a dimension to cellular complexity and benefit cells in several ways. This phenomenon occurs in both eukaryotes and prokaryotes. For example, moonlighting proteins from the glycolytic pathway or TCA cycle can play a role in bacterial pathogenesis by either acting as proteins secreted in a conventional pathway and/or as cell surface components that facilitate adhesion or adherence. This review outlines the multifunctionality exhibited by many Paracoccidioides spp. enzymes, including aconitase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate lyase, malate synthase, triose phosphate isomerase, fumarase, and enolase. We discuss the roles that moonlighting activities play in the virulence characteristics of this fungus and several other human pathogens during their interactions with the host.Frontiers in microbiology. 01/2014; 5:719.
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ABSTRACT: Epithelial cells lining the urinary tract secrete urinary exosomes (40-100nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and non-invasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date while a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique non-modified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes.Molecular & Cellular Proteomics 12/2014; · 7.25 Impact Factor
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ABSTRACT: Background Exosomes are membranous nanovesicles secreted into the extracellular milieu by diverse cell types. Exosomes facilitate intercellular communication, modulate cellular pheno/genotype, and regulate microbial pathogenesis. Although human semen contains exosomes, their role in regulating infection of viruses that are sexually transmitted remains unknown. In this study, we used semen exosomes purified from healthy human donors to evaluate the role of exosomes on the infectivity of different strains of HIV-1 in a variety of cell lines.ResultsWe show that human semen contains a heterologous population of exosomes, enriched in mRNA encoding tetraspanin exosomal markers and various antiviral factors. Semen exosomes are internalized by recipient cells irrespective of cell type and upon internalization, inhibit replication of a broad array of HIV-1 strains. Remarkably, the anti-HIV-1 activity of semen exosomes is specific to retroviruses because semen exosomes blocked replication of the murine AIDS (mAIDS) virus complex (LP-BM5). However, exosomes from blood had no effect on HIV-1 or LP-BM5 replication. Additionally, semen and blood exosomes had no effect on replication of herpes simplex virus; types 1 and 2 (HSV1 and HSV2). Mechanistic studies indicate that semen exosomes exert a post-entry block on HIV-1 replication by orchestrating deleterious effects on particle-associated reverse transcriptase activity and infectivity.Conclusions These illuminating findings i) improved our knowledge of the cargo of semen exosomes, ii) revealed that semen exosomes possess anti-retroviral activity, and iii) suggest that semen exosome-mediated inhibition of HIV-1 replication may provide novel opportunities for the development of new therapeutics for HIV-1.Retrovirology 11/2014; 11(1):102. · 4.77 Impact Factor
Immunology Letters 107 (2006) 102–108
Exosomes: From biogenesis and secretion to biological function
Sascha Kellera, Michael P. Sandersona, Alexander Stoecka,b, Peter Altevogta,∗
aGerman Cancer Research Center (DKFZ), Tumor Immunology Program, D010/TP3, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
bDepartments of Pediatrics and Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, United States
Received 13 September 2006; accepted 21 September 2006
Available online 17 October 2006
Exosomes are small microvesicles that are released from late endosomal compartments of cultured cells. Recent work has shown that exosome-
like vesicles are also found in many body fluids such as blood, urine, ascites and amnionic fluid. Although the biological function of exosomes is
far from being fully understood, exosomes may have general importance in cell biology and immunology. The present review aims to address some
of the facets of exosome research with particular emphasis on the immunologist’s perspective: (i) exosomes as a novel platform for the ectodomain
shedding of membrane proteins by ADAMs and (ii) recent findings on the role of exosomes in tumor biology and immune regulation.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Exosomes; Membrane vesicles
Exosomes are small microvesicles released from cells, and
have been the subject of intensive research in recent years.
Originally described as a mechanism for the removal of cell
surface molecules in reticulocytes, the pioneering work of
several labs showed the importance of exosomes for gen-
eral cell biology and in particular for the immune system.
The reader is also encouraged to refer to other excellent
reviews on exosomes biology that have recently been published
1.1. The endosomal pathway, exosome biogenesis and
Eukaryotic cells stay in contact with the environment by
receiving signals such as cytokines or chemokines, the uptake
of nutrients and the secretion of proteins into the extracellular
space. For uptake and secretion, each cell has a complex net-
work of membranes inside the cell. Using these compartments,
ment (endocytosis) but also release newly-synthesized proteins
or carbohydrates (exocytosis).
∗Corresponding author. Tel.: +49 6221 423714; fax: +49 6221 423791.
E-mail address: P.Altevogt@dkfz.de (P. Altevogt).
There are different mechanisms by which cells release pro-
teins into the extracellular space. The most common process
is the release of large biomolecules through the plasma mem-
exocytosis has a regulatory or signaling function. According to
the mechanism of release, exocytosis can be divided into two
different modes: (1) constitutive (non-calcium-triggered) or (2)
regulated (calcium-triggered) exocytosis. Constitutive exocyto-
into the plasma membrane after fusion with transport vesicles.
Regulated exocytosis is particularly important in neurological
signaling, where synaptic vesicles fuse with the membrane at
the synaptic cleft and induce nerve impulses [3,4].
Fusion of multivesicular bodies (MVBs) with the plasma
membrane and the subsequent release of their cargo represents
another mechanism of exocytosis. Since the development of
these membrane vesicles has an endocytic origin, this mech-
anism is a secretion process of the endosomal system. Other
early endosomes, late endosomes and lysosomes. Endocytic
vesicles arise through clathrin- or non-clathrin-mediated endo-
cytosis at the plasma membrane and are transported to early
endosomes. Late endosomes develop from early endosomes by
to fuse with vesicles or more generally with other membranes
0165-2478/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
S. Keller et al. / Immunology Letters 107 (2006) 102–108
. These different vesicles can be distinguished by their phys-
ical shape and cellular location. In particular, early endosomes
display a tubular appearance and are located at the outer mar-
gin of the cell, whereas late endosomes are spherical in shape
and are located close to the nucleus. The key step in the forma-
tion of MVBs from late endosomes is reversed budding. During
luminal vesicles .
1.1.2. Fates of vesicles within MVBs: degradation versus
The fusion of a MVB with the lysosome results in the degra-
allows the cell to remove transmembrane proteins as well as
excessive membranes [7,8]. The degradation of transmembrane
proteins plays an important role in the down-regulation of acti-
factor receptors become internalized by ligand-induced endo-
cytosis and are sorted into the luminal vesicles of the MVB
to then undergo further degradation in the lysosome. Normal
internalization without degradation is in some cases insuffi-
cient in diminishing signaling by the receptors. As depicted in
C-terminal phosphorylated docking sites and kinase domain of
the activated epidermal growth factor receptor (EGFR/ErbB1)
retain access to the cytosol where further signal transduction
can occur. However, sorting into MVBs isolates the activated
receptors from the cytosol and thereby from binding partners.
Interestingly, mice carrying a mutation resulting in defective
sorting of the EGFR into MVBs have a higher risk of develop-
ing tumors [9,10].
In dendritic cells (Fig. 1B), MVBs play a critical role as
a storage compartment for MHC class II molecules and their
associated invariant chain [11,12]. In this case, the MVB com-
partment is called the MHC class II compartment (MIIC). Fol-
lowing internalization of antigen by dendritic cells, the invari-
ant chain is removed and the MHC complex becomes loaded
with the antigen peptide. These internal vesicles of the MIIC
then fuse with the plasma membrane resulting in the presen-
tation of antigen-loaded MHC class II molecules on the cell
MVBs can also fuse with the plasma membrane leading
to the release of the internal vesicles into the extracellular
space (Fig. 1C). The released vesicles are then called exosomes.
Many cell types release exosomes via this mechanism includ-
ing hematopoietic cells, reticulocytes, B- and T-lymphocytes,
dendritic cells, mast cells, platelets, intestinal epithelial cells,
astrocytes, neurons and tumor cells [13–21]. Depending on
their origin, exosomes have previously been named dexosomes
Fig. 1. Different fates and functions of internalized vesicles. (A) Lysosomal degradation: some cell surface receptors such as the EGFR are internalized following
ligand binding and activation. Degradation of the receptor following trafficking to lysosomes functions to down-regulate receptor signaling. (B) MHC class II storage
compartment: antigens taken up into vesicles are degraded into shorter peptides which bind to MHC class II molecules in the MHC class II storage compartment
(MCII). After delivery of the loaded MHC complexes to the cell surface, they can be recognized by CD4+ T cells. (C) Release of exosomes: multivesicular bodies
can fuse with the plasma membrane and release internal vesicles (exosomes) into the extracellular environment.
S. Keller et al. / Immunology Letters 107 (2006) 102–108
(dendritic cell-derived exosomes) or texosomes (T-cell exo-
somes or tumor exosomes).
1.1.3. Sorting into the MVB and exosomes
Only very little is know about the sorting signals which
are responsible for the sorting of proteins into vesicles within
MVBs, which can be subsequently released as exosomes. As
mentioned earlier, binding of a ligand to cell surface receptors
results in receptor activation and initiation of signal transduc-
tion pathways. The fate of different activated receptors can be
highly variable. Some receptors pass multiple cycles of uptake
and recycling to the plasma membrane, whilst others, which
are destined for degradation, are directly transported to lyso-
somes. In the case of most cellular transport mechanisms, such
as nuclear translocation, proteins intended for a specific com-
partment display a characteristic amino acid sequence, which
acts as a sorting signal . For the recruitment of proteins into
MVBs, there is currently no common sorting signal known for
and trafficking of proteins to MVBs and exosomes.
For the EGF receptor, a point-mutation has been identified
romyces cerevisiae, ubiquitinylation of the G-protein coupled
receptor (GPCR) Ste2 leads to trafficking into MVBs and sub-
ubiquitin polypeptide is processed by a cascade of enzymes and
becomes ligated to lysine residues of substrate proteins. The
ligation of a single ubiquitin moiety (mono-ubiquitinylation)
acts as a signal for endocytosis and the delivery into MVBs.
However, the attachment of multiple ubiquitin chains (poly-
ubiquitinylation) directs proteins for degradation in the protea-
via ubiquitin-triggered internalization. The formation of MVBs
relies on ubiquitin-binding proteins [26,27]. The endosomal
sorting complex required for transport (ESCRT) recognizes the
ubiquitinylated cargo via Vps-27. Vps-27 then recruits another
ESCRT complex and Tsg-101, which activate AIP/Alix. This
complex drives the cargo into the budding vesicles. Although
mono-ubiquitinylation triggers the uptake into MVBs, not all
proteins in exosomes are ubiquitinylated. It appears that there is
also a passive mechanism involved in protein sorting to MVBs.
The responsible signals for this processes are in some cases the
presence of tetraspanin enriched  or cholesterol enriched
(=lipid rafts) membrane microdomains .
1.2. Exosomal structure and integral constituents
Exosomes are classically defined as membranous vesicles
with a diameter of 30–100nm. Many groups have performed
proteomic analysis of vesicles derived from cell lines or body
fluids such as urine, blood and ascites. Such analyses have
shown that all mammalian exosomes share some common char-
acteristics such as structure (bilipidic layer), size, density and
overall protein composition. Some proteins are located on the
surface or in the lumen of nearly all exosomes (exosomal mark-
ers). Notably, these include cytoplasmic proteins such as tubu-
lin, actin, actin-binding proteins, annexins and Rab proteins as
well as molecules responsible for signal transduction (protein
kinases, heterotrimeric G-proteins) [30–32]. Most exosomes
commonly associated with exosomes is the tetraspanins includ-
ing CD9, CD63, CD81 and CD82 [36–38]. Conversely, other
exosomal proteins directly represent the proteome of the source
cells. For example, analysis of urinary vesicles showed a link
the urogenital tract . Meanwhile, urinary vesicles have been
examined for their potential use in the detection of malignancy-
associated proteins and from these analysis it was concluded
. As exosomes are also found in serum and ascites fluids of
tumor patients, it is possible that exosome analysis may eventu-
ally become important for diagnosis and biomarker analysis. In
in exosomes . Consistent with their endosomal origin, there
are typically no proteins of the nucleus, mitochondria, or endo-
plasmic reticulum detectable in exosomes . In contrast, all
exosomal proteins are typically found in the cell cytosol or at
the plasma membrane.
1.3. Biological functions of exosomes and other secreted
In contrast to the fate of proteins trafficked for degradation to
entities which are important for a variety of pathways. Some
examples of roles for exosomes are discussed below.
1.3.1. Morphogen signaling
Exosomes and other cell-derived soluble vesicle compart-
ments can themselves act as biologically active signals. For
example, in developmental biology, morphogens play an essen-
spread through the adjacent tissue at different concentrations.
Pattern formation in developing Drosophila tissues occurs in
less and Hedgehog [42–44]. Interestingly, Wingless is tightly
[45,46]. It has been reported that argosomes arise by a mech-
anism similar to the formation of exosomes from MVBs. The
tosis events. Thereby, the main contingent of argosomes travels
thorough tissues and is found in endosomes, whilst few argo-
1.3.2. Exosomes as immunological mediators
Exosomes display a wide variety of immunostimulatory
properties. For example, exosomes secreted by Eppstein-Barr
virus (EBV)-transformed B cells are able to stimulate CD4+
T cells in an antigenic-specific manner . Meanwhile,
S. Keller et al. / Immunology Letters 107 (2006) 102–108
exosomes produced by mouse dendritic cells pulsed with tumor
peptide are able to mediate the rejection of established tumors
[13,47]. These antitumor effects were antigen-specific and were
associated with the activity of T cells. Direct stimulation of T
cells by membrane vesicles from antigen-presenting cells has
also been reported . Conversely, it has been suggested that
intestinal epithelial cells, T-cell tumors and melanoma cells
can secrete exosomes capable of inducing antigen-specific
tolerance and FasL-mediated T-cell apoptosis [49,50].
A recent study has shown a role for exosomes in the mod-
ulation of T-cell signaling in pregnancy . Exosomes from
the serum of pregnant women could suppress the expression
of important T-cell signaling components including CD3-? and
FasL and a striking difference was noted between women deliv-
ering at term and those delivering pre-term. Marker analysis
by which the placenta promotes a state of immune privilege.
Exosomes were also shown to play a role in the control
of tumor growth . Pretreatment of mice with exosomes
derived from murine mammary carcinomas augmented subse-
quent tumor growth by inhibiting the cytolytic activity of NK
cells. On the molecular level, tumor exosomes diminished lev-
els of perforin in NK cells, a molecule that is essential for target
cell lysis. It was shown that exosomes are taken up and remain
stable in NK cells. Meanwhile, mRNA levels of perforin were
not affected by exosomes, suggesting a post-translational regu-
latory mechanism. One possibility could be that perforin, stored
in granula, becomes degraded by exosomes that have entered
the NK cell.
pulsed with tumor-specific peptides can active the immune sys-
tem and are a valuable source of material for immunotherapy.
Clinical trials are currently being conducted to assess the safety
and efficacy of anti-tumor vaccines using exosomes . How-
ever, the finding that exosomes mediate positive and negative
immune regulatory functions suggests that the application of
exosomes in immunotherapy must be careful examined prior to
onset of further clinical applications.
1.3.3. Ectodomain shedding of proteins in the MVB and
Transmembrane proteins in many cases can undergo cell
surface cleavage to generate a soluble form of the molecule
(Fig. 2A). This has been demonstrated for a large number of
molecules including growth factors and receptors, adhesion
soluble forms often exhibit alternate roles to the transmem-
brane form. For example, the transmembrane form of heparin-
binding epidermal-like growth factor (HB-EGF) binds via its
extracellular ectodomain to diphtheria toxin and the diphthe-
ria toxin receptor-associated protein (DRAP27/CD9) . In
addition, the intracellular C-terminus of transmembrane HB-
EGF binds the anti-apoptotic protein BAG-1 and this affects
cell morphology, adhesion and resistance to apoptosis .
uble form of HB-EGF binds the EGFR to mediate a variety of
Fig. 2. Exosomes as a novel platform for ADAM-mediated cleavage. (A) Cleavage of cell surface proteins such as L1 and CD44 can occur at the cell surface. A
second pathway of cleavage is initiated by the endocytosis of both ADAM proteases and cell surface substrates. During vesicles maturation, additional cargo is
derived from the golgi and the trans golgi network (TGN). (B) Proteolysis of substrate transmembrane molecules by ADAMs can also occur in MVBs. The resulting
cleaved/soluble forms are secreted by fusion with the plasma membrane.
S. Keller et al. / Immunology Letters 107 (2006) 102–108
zinc-binding metalloproteases such as the ADAM (a disintegrin
and metalloprotease), MMP (matrix metalloprotease) and MT-
MMP (membrane-type matrix metalloprotease) families .
and we have demonstrated that the two most widely character-
ized ectodomain sheddases ADAM10 and ADAM17 (TACE)
membrane molecules .
The majority of ectodomain shedding reports have sug-
gested that proteolysis of transmembrane molecules occurs at
the cell surface. However, in the case of several molecules,
ular compartments in MVBs and within secreted exosomes. For
example, we have shown that the L1 adhesion molecule and
CD44 undergo proteolytic ectodomain shedding in MVBs by
ADAM10 [59–61]. The cleaved/soluble ectodomains of L1 and
CD44 can then be directly released from the cell via exocytosis.
are released from the cell within exosomes and can undergo fur-
ther ADAM10-mediated cleavage to generate soluble forms of
each molecule (Fig. 2B). The notion for a role of exosomes
in ectodomain shedding and protein secretion is supported by
similar findings for the transmembrane proteins CD46 and the
tumor necrosis factor receptor 1 (TNFR1). Ovarian adenocar-
cinoma cell lines release full-length transmembrane CD46 in
vesicles . Vesicle-associated CD46 can then become fur-
ther processed by metalloproteases to generate a soluble form.
Meanwhile, TNFR1 is released from vascular endothelial cells
Most fascinatingly, both CD46 and TNFR1 maintain their bio-
logical activity on the exosome surface with regards to ligand
binding. These findings suggest a crucial role for exosomes in
the biological function and ectodomain shedding of a variety of
Interestingly, exosome secretion and ADAM-mediated
ectodomain shedding may be tightly related processes. Stimuli
loprotease activators (e.g. 4-aminophenylmercuric acetate) and
agents which stimulate cholesterol extraction from the plasma
membrane (e.g. methyl-?-cyclodextrin) are also stimulators
of exosome release . In addition, inhibitors of ADAM-
metalloproteases block exosome formation . This raises
the possibility that metalloproteases such as ADAM10 and
ADAM17, which are present in endosomes and exosomes, may
play a direct role in exosome formation.
sis of exosomes has been demonstrated using in vitro cell lines,
many questions regarding the biological role of exosomes in
complex cellular systems remain to be addressed. It is clear that
exosome-like microvesicles are present in body fluids such as
blood, urine, amnionic fluid ascites and pleural effusions under
healthy and disease conditions. However, the origin of these
exosomes and their destination for stimulation of distal cells
remains unclear. It has been demonstrated that exosomes can
be taken up by other cells. However, whether this establishes a
unanswered question. The release and subsequent cleavage of
transmembrane protein in microvesicles may be a novel mech-
anism of secretion that is distinct from the classical exocytosis
pathway. It is presently unclear how exosomal secretion is reg-
ulated; however, recent work has shown that the p53 protein
and the p53 regulated protein TSAP6 may be involved in this
process . Meanwhile, it is worth mentioning that in certain
disease conditions, exosomes may play regulatory functions.
This is highlighted by the recent finding that ?-amyloid pep-
tides, associated with Alzheimer’s disease (AD), are release
in association with exosomes and that exosomal proteins were
found to accumulate in the plaques of AD patients brains .
In addition, prions are released from cells in association with
exosomes and trafficking within the body functions as an infec-
tious route for propagation of disease [66,67]. The release of
exosomes from tumor cells may also be a novel mechanism
for chemoresistance. For example, enhanced exosomal export
of cisplatin was observed in drug-resistant human ovarian carci-
the studies mentioned in this review highlight multiple roles for
secreted exosomes in a range of biological settings including
development, immunology and cancer. Whilst functional roles
of exosomes are only recently becoming clear, future investiga-
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