Báo cáo Y học: Secretion of a peripheral membrane protein, MFG-E8, as a complex with membrane vesicles A possible role in membrane secretion pptx

Secretion of a peripheral membrane protein, MFG-E8,
as a complex with membrane vesicles
A possible role in membrane secretion
Kenji Oshima
1
, Naohito Aoki
1
, Takeo Kato
2
, Ken Kitajima
1
and Tsukasa Matsuda
1
1
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan;
2
Food Research Institute, Aichi Prefectural
Government, Nagoya, Japan
MFG-E8 (milk fat g lobule-EGF factor 8) is a p eripheral
membrane glycoprotein, which is expressed abundantly in
lactating mammary glands and is secreted in association with
fat globules. This protein consists of two-repeated EGF-l ike
domains, a mucin-like domain and two-repeated discoidin-
like domains ( C-domains), a nd contains an integrin-binding
motif (RGD sequence) in the EGF-like domain. To clarify
the role o f each domain on the peripheral association with
the cell membrane, several domain-deletion mutants of
MFG-E8 were expressed in COS-7 cells. The immunofluo-
rescent s taining o f i ntracellular and cell-surface proteins and
biochemical analyses of cell-surface-biotinylated and secre-
ted proteins demonstrated that both of the two C-domains

were required f or the membrane a ssociation. During t he
course of these studies for domain functions, MFG-E8, but
not C-domain deletion mutants, was shown to be s ecreted as
membrane vesicle complexes. By size-exclusion chromato-
graphy and ultracentrifugation analyses, the c omplexes were
characterized to have a h igh-molecular mass, low d ensity
and higher s edimentation velocity a nd to be detergent-
sensitive. Not only such a exogenously expressed MFG-E8
but also that endogenously expressed in a mammary epi-
thelial cell line, COM MA-1D, was secreted as the membrane
vesicle-like complex. S canning electron microscopic a nalyses
revealed that MFG-E8 was secreted into the culture medium
in association with small membrane vesicles w ith a size from
100 to 200 nm in diameter. Furthermore, the expression of
MFG-E8 increased the number of these membrane vesicle
secreted into the culture medium. These results suggest a
possible role o f MFG-E8 in the membrane vesicle secretion,
such as budding or shedding of plasma membran e (micro-
vesicles) and exocytosis of endocytic multivesicular bodies
(exosomes).
Keywords: MFG-E8; membrane secretion; exosome; per-
ipheral membrane protein; milk fat globule membrane.
MFG-E8 (milk fat globule-EGF factor 8) was cloned and
characterized as mouse milk 53- and 66-kDa glycoproteins
peripherally associated with the membrane surrounding the
lipid droplets and being referred to as milk fat globule
membrane (MFGM) [1]. MFG-E8 consists of two repeated
EGF-like domains on the N-terminal side and of two
repeated C (discoidin-like) domains homologous to the C1
and C 2 domains of blood coagulation factors V and VIII.

Orthologous proteins have been isolated in bovine
(MGP57/53 or PAS-6/7) [2,3], human (BA46 or lacta-
dherin) [4,5] and rat (rAGS) [6].
Though the expression of MFG-E8 is upregulated in
lactating mammary gland, MFG-E8 has also been detected
in various other tissues, including brain, lung, heart, kidney
and spleen in some mammals such as mouse, human and
bovine [7–9]. The mouse and bovine MFG-E8 proteins
expressed in mammary gland were shown t o be composed
of two isoforms [ 3,9]. In mouse, a Pro/Thr-rich domain is
inserted possibly by a mammary gland-specific alternative
splicing between EGF-like and C-domains, resulting in the
production of a long form of MFG-E8 (MFG-E8-L) in the
lactating mammary gland. In contrast, a short form ( MFG-
E8-S) lacking the Pro/Thr-rich domain is ubiquitously
expressed in various tissues [9].
The second EGF-like domain of MFG-E8 contains an
integrin-binding Arg-Gly-Asp (RGD) sequence motif [10],
which is conserved in all known MFG-E8 sequences of
several species and binds to some integrins. The avb5
integrin was affinity-purified from lactating bovine udder
extracts by using its specific binding to bovine milk
MFG-E8 [7], and human and bovine MFG-E8 proteins
promoted cell adhesion through avb3andavb5 i ntegrins
[11, 12]. Although MFG-E8 contains no apparent hydro-
phobic transmembrane regions, MFG-E8 has been shown
to be a peripheral membrane protein and bind directly to
the MFGM and cell membrane [7,13–15]. Both the native
and recombinant MFG-E8 proteins bind in vitro to anionic
phospholipids, especially phosphatidylserine (PtdSer)

[7,12,16]. This PtdSer-binding of MFG-E8 has been
reported t o d epend only on the second C-domain
(C2-domain), but not the first C-domain (C1-domain),
in the same manner a s that o f blood coagulation factors
V and VIII [17–19].
Correspondence to T. Matsuda, Department of Applied Molecular
Biosciences, Graduate School of Bioagricultural Sciences, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya 464-8601, J apan.
Fax: + 81 52 789 4128, Tel.: + 81 52 789 4129,
E-mail:
Abbreviations: MFGM, milk fa t g lobule membr ane; DM EM,
Dulbecco’s modified Eagle’s serum; DAPI, 4¢,6-diamidine-2-phenyl-
indole-dihydrochloride; ECL, enhanced chemiluminescence; MVBs,
endocytic multivesicular bodies; GST, glutathione S-transferase.
(Received 19 September 2001, revised 17 December 2001, accepted 2
January 2002)
Eur. J. Biochem. 269, 1209–1218 (2002) Ó FEBS 2002
Recently, MFG-E8 was d etected as the major component
of the secretory membrane v esicle (exosome) secreted by a
murine dendritic cell line (D1) [20]. Furthermore, a glioma
cell line (C6) has a lso been shown to secrete MFG-E8 into
the culture media [21]. MFG-E8 is also detected extracell-
ularly in embryonic gonad [22] and in sera of patients with
breast tumor metastasis [23]. Thus, the results reported so
far suggest that MFG-E8 secreted extracellularly, at least in
some occasions, despite the membrane associated nature of
MFG-E8. Aims of the present study are to elucidate cellular
and extracellular distribution of MFG-E8 expressed in
cultured mammalian cells and to identify domain(s)
responsible for the membrane association and/or secretion.

By using transformed COS-7 cells as well as a mammary
epithelial cell line, COMMA-1D expressing MFG-E8, we
have found that MFG-E8 exists not only on the cell surface
but also in association with unc haracterized membrane
vesicles secreted into culture medium. The expression of
several domain-deletion mutants of MFG-E8 suggests
contribution of the C2 domain to the association with
PtdSer and plasma membrane and subcontribution of the
C1 domain to the association with plasma membrane.
A possible role of MFG-E8 in the vesicular secretion is also
discussed.
EXPERIMENTAL PROCEDURES
Cell culture
A mouse m ammary epithelial cell line, COMMA-1D, and a
monkey kidney cell line, COS-7 w ere c ulture d i n Dulbecco’s
modified Eagle’s medium (DMEM, Sigma) containing
10% heat-inactivated fetal bovine serum, penicillin at
100 U ÆmL
)1
, streptomycin at 100 lgÆmL
)1
at 37 °C under
humidified 5% CO
2
and 95% air.
Construction of expression plasmids
and gene transfection
MFG-E8-L and -S expression plasmids were generated as
described previously [9]. A truncated MFG-E8-L cDNA
that lacks a region encoding the C1 domain (amino acids

147–306) was constructed as follows. The cDNA fragments
upstream a nd downstream of t he C1 domain were amplified
by PCR using the MFG-E8-L expression p lasmid as a
template w ith p rimer s ets c ontaining an XbaI site a s follows:
5¢-ATGCAGGTCTCCCGTGTGC-3¢,5¢-AT
TCTAGAG
GCTAGGTTGTTGGAAAG-3¢,5¢-AT
TCTAGAGGAT
GTCTTGAGCCCCTG-3¢ and 5¢-TTCTCGAGCAGGA
CTGAGCATTAACAG-3¢. The two DNA fragments were
ligated at the XbaI site and inserted into a cloning vector
pBluescript KS(+) (Stratagene). As a result of the ligation,
the domain to be deleted was replaced by two amino acids,
serine and a rginine, which we re translated fro m the XbaI-
site sequence T CTAGA. T he c DNA lacking the C1 domain
was then amplified by PCR from the cloned plasmid
with primers containing an EcoRI site at the 5¢ end
(5¢-TA
GAATTCCACCATGCAGGTCTCCCGT-3¢)and
an EcoRV site at the 3¢ end (5¢-CA
GATATCTTAACAGC
CCAGCAGCTC-3¢). The cDNA lacking the C2 domain
(amino acids 307–463) was created by PCR directly from
the MFG-E8-L expression plasmid w ith primers containing
an EcoRI site at the 5¢ end as same above and an EcoRV
site at the 3¢ end (5¢-CAGATATCTTAGTGCAACTCAC
AGCC-3¢). The two PCR products were cloned into a
mammalian expression vector, pEF1/Myc-His C (Invitro-
gen), at EcoRI and EcoRV sites, respectively, and were
checked by sequencing for PCR errors.

COS-7 cells were seeded at a d ensity of 2.5 · 10
5
cells per
60-mm dish, an d grown overnight in DMEM containing
10% fetal bovine serum. The cells were transfected with the
plasmid DNA by the calcium phosphate-DNA precipita-
tion method [24]. After incubation under 3 % CO
2
and 97%
air for 18 h, the transfected cells were washed with NaCl/P
i
and cultured under humidified 5% CO
2
and 95% air.
Immunofluorescence staining
COS-7 cells were cultu red on cover glasses and transfected
with MFG-E8s expression plasmids as described above.
After being cultured in DMEM containing 10% fetal
bovine serum fo r 24 h, cells were washed three times with
NaCl/P
i
andfixedwith3%paraformaldehydeinNaCl/P
i
for 8 min for extracellular s taining or with methanol chilled
at )20 °C for 5 min for intracellular staining. After blocking
with NaCl/P
i
containing 2% BSA (blocking solution) for
30 min, the specimens were incubated for 60 min with the
rabbit a ntiserum raised against the recombinant glutathione

S-transferase (GST)–MFG-E8 fusion protein [8] diluted
1 : 150 in blocking solution and then incubated for 30 min
with the secondary antibody, FITC-labeled goat anti-
(rabbit IgG) I g ( ICN/Cappel). S amples were then incubated
for 1 5 min wit h 4¢,6-diamidine-2-phenylindole-dihydrochlo-
ride (DAPI) (Roche Molecular B iochemicals) ( 1 lgÆmL
)1
NaCl/P
i
) a nd washed three times with NaCl/P
i
.Imageswere
acquired by using a fluorescence microscope (Olympus).
SDS/PAGE and Western blotting
The transfected cells were cultured in serum-free DMEM
for 24 h and then lysed with Ôlysis bufferÕ containing 50 m
M
Hepes (pH 7.5), 150 m
M
NaCl, 10% glycerol, 1% Triton
X-100, 5 m
M
EDTA, 1 m
M
phenylmethanesulfonyl fluoride
and 10 lgÆmL
)1
leupeptin. The culture supernatant of the
transfected COS-7 cells was concentrated to one-sixtieth of
its original volume by centrifugal filtration through the M

r
10 000 cut-off membrane (Amicon). Proteins in the cell
lysate and the culture medium were separated by SDS/
PAGE (10% acrylamide gel) and electrophoretically trans-
ferred to the membrane Immobilon-P (Millipore). The
membrane was blocked and then sequentially incubated
with the rabbit anti-(GST–MFG-E8) serum and peroxi-
dase-conjugated goat anti-(rabbit IgG) Ig. The protein
bands probed with the peroxidase-labeled antibody were
visualized with an enhanced chemiluminescence (ECL)
detection kit (Amersham Pharmacia Biotech).
Cell surface biotinylation
COS-7 cells were plate d onto six-well polystyrene plates at a
density o f 1 · 10
5
cells per well and transfected with the
MFG-E8 expression plasmids as described above. After
incubation in DMEM containing 10% fetal bovine
serum for 48 h, the cells were washed three times with
cold NaCl/P
i
and incubated at 4 °C for 30 min in the
presence of 0.5 mgÆmL
)1
Sulfo-N-hydroxysulfosuccinimide-
1210 K. Oshima et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Biotin (Pierce). After nonreacted biotin was quenched with
serum-free DMEM at 4 °C for 5 min, cells were washed
three t imes with NaCl/P
i

and t hen lysed with the lysis buffer.
Streptavidin–Sepharose (Amersham Pharmacia Biotech)
was added to the cell lysate and incubated overnight at
4 °C. Proteins bound to the S epharose were precipitated
by centrifugation and washed extensively with a buffer
containing 50 m
M
Hepes (pH 7.5), 150 m
M
NaCl,
10% glycerol, 0.1% Triton X100 and then subjected to
SDS/PAGE followed by Western blotting.
Size-exclusion chromatography of the secreted MFG-E8
Culture supernatants from the transfected COS-7 cells
(8 · 10
6
cells) grown in serum-free DMEM for 24 h were
concentrated as described above. The concentrated super-
natants (500 lL) were subjected to the size-exclusion
chromatography using a Sephacryl S-300 column
(0.9 · 60 cm), equilibrated with NaCl/P
i
. The elution
profiles of MFG-E8 and its mutants were monitored by
ELISA. ELISA plate was coated directly with each fraction,
and the antigens were detected by using the rabbit a nti-
(GST–MFG-E8) serum and peroxidase-conjugated goat
anti-(rabbit IgG) Ig as described previously [25].
Ultracentrifugation
Culture supernatants from COMMA-1D cells (8 · 10

6
cells) and t he transfected COS-7 ce lls (4 · 10
6
cells) were
prepared and concentrated as described above. Aliquots
(500 lL) of the concentrated samples were clarified by
sequential centrifugation at 1200 g (10 min) and 10 000 g
(30 min) to eliminate cells and debris. In the experiment to
examine the effect of detergent, 50 lL of 10% Triton X-100
was then added to the supernatants of the centrifugation,
followed b y t he incubation on ice for 10 min. These samples
with or without Triton X-100 were ultracentrifuged at
100 000 g for 1 h at 4 °C. The resulting supernatants were
recovered, while the pellets were resuspended in 100 lLof
NaCl/P
i
containing 0.01% sodium azide and 10 lgÆmL
)1
leupeptin. The presence of MFG-E8 was determined by
Western blotting for both of the supernatants and pellets.
Sucrose density-gradient ultracentrifugation
Culture supernatants from the transfected COS-7 cells
(8 · 10
6
cells) were prepared and concentrated as described
above. Concentrated samples (500 lL) were mixed with
2.5 vol. of buffer A [85% (w/v) sucrose in 10 m
M
Tris/HCl
(pH 7.5) containing 150 m

M
NaCl and 5 m
M
EDTA], and
placed in centrifuge tubes. The mixtures were layered
successively with 4 mL of 60% (w/v), 3 mL of 30% (w/v)
and 1 mL of 5% (w/v) sucrose in buffer A, and centrifuged
at 200 000 g for 18 h at 4 °C ( Beckman L-70K centrifuge,
SW41 Ti rotor). The fractions with different densities were
collected with 1 mL portions from the top to the bottom of
the tube. Each fraction was directly subjected to SDS/
PAGE followed by Western blotting.
Phospholipid-binding assay by ELISA
The ELISA for MFG-E8 binding to solid-phase phospho-
lipid was performed as described previously [7].
L
-a-phosphatidyl-
L
-serine (Sigma) in methanol
(10 lgÆmL
)1
) was added to a micro well plate (Nunc)
(30 lLÆwell
)1
) followed by drying a t 37 °C. The plate was
washed three t imes between all subsequent steps w ith NaCl/
Tris containing 0.05% Tween-20. The p late was block ed
with 200 lL of NaCl/Tris containing 0.05% (w/v) gelatine
(blocking buffer). Culture supernatants of the t ransfected
COS-7 cells were concentrated to one-fourth of its original

volume. Appropriate amounts of total proteins in the
supernatants were then diluted in 50 lL of blocking buffer,
and were added per well, followed b y incubation at 4 °C
overnight. The plate was then incubated with anti-(GST–
MFG-E8) serum and p eroxidase-labeled goat anti-(rabbit
IgG) Ig as the secondary antibody, and peroxidase activity
was measured.
Scanning electron microscopy
Samples for scanning electron microscopy analysis were
prepared essentially as previously described for microvesi-
cles [ 26]. Culture supernatants of the t ransfected COS-7 cells
(2 · 10
6
cells) cultured in serum-free DMEM for 48 h were
centrifuged a t 1 0 000 g for 30 min to eliminate cells and
debris. The supern atants were then centrifuged at 200 000 g
for 1.5 h a t 4 °C. The pellets were r esuspended in 100 lLof
NaCl/Tris with 0.01% sodium azide and 10 lgÆmL
)1
leupeptin. The suspended samples were placed onto micro-
scope glass slides, previously treated w ith poly
L
-lysine
(Sigma) for 30 min, and then fixed w ith 1% OsO
4
for
2 h. The samples were dehydrated in a series of ethanol
(50–100%), c ritical-point d ried in a C O
2
system. A fter being

platinum/palladium-coated in a sp attering devise, the speci-
mens were observed with a scanning electron microscope
(JSM-820, Japan Electron Optics Laboratory).
RESULTS
Cellular and extracellular distribution of MFG-E8
and its domain deletion mutants expressed in COS-7 cells
To investigate cell surface distribution of MFG-E8 and
contribution of each domain to the cellular localization,
several domain-deletion mutant genes for MFG-E8 was
constructed (Fig. 1) and transiently expressed in COS-7
cells. The transfected cells were fixed, and the cell surface
and intracellular MFG-E8 proteins were detected by
indirect immunofluorescence staining using the antibody
specific for MFG-E8. As s hown in F ig. 2, under a
nonpermeable condition the two wild-type MFG-E8
proteins (MFG-E8-L and -S) were detected as many dots
on the surface of transfected cells, whereas no signal was
detected for the two C-domain deletion mutants (DC1 and
DC2). Under permeab le conditions, however, all of MFG-
E8 and deletion mutants were clearly detected in cyto-
plasm. No signal was detected under permeable or
nonpermeable conditions for an empty-vector (mock)
transfectant. Thus, MFG-E8, expresse d in COS-7 cells,
localized on the cell surface and both of the two
C-domains w ere indispensable for such a cell surface
localization of MFG-E8. A possibility that cytosolic
MFG-E8 was stained under t he nonpermeable condition
could be excluded because DC1 and DC2 were not
detected under t he same condition.
Ó FEBS 2002 MFG-E8: a component of secreted membrane vesicles (Eur. J. Biochem. 269) 1211

As the expression and l ocalization of MFG-E8 and its
deletion mutants in COS-7 cells was revealed immunocyto-
chemically, biochemical analyses including cell surface
biotinylation were done subsequently for both of the
transfected cells and their culture supernatants. As shown
in Fig. 3, MFG-E8-L and -S were clearly labeled by the cell
surface b iotinylation, confirming their existence o n the cell
surface. In contrast, only w eak or a lmost no b ands were
detected for the C-domain deletion mutants, indicating that
they did not retain on the cell surface. Whe n the cu lture
supernatants were analyzed, considerable amounts of the
C-domain deletion mutan ts were found to be secreted into
the culture medium. Furthermore, MFG-E8-L and -S were
detected in cu lture supernatant, indicating that they were
not only plasma-membrane-associated but also secreted.
While three s ize-variants (66, 56 and51 kDa) f or M FG-E8-L
were detec ted in the total ce ll lysate and cell-surface
biotinylated proteins, s ecreted MFG-E8-L was only a single
band of 66 kDa. On the other hand, the molecular mass of
MFG-E8-S was 51 kDa regardless its secretory o r cellu lar
form. Both of DC1 (47 kDa) and DC2 (43 kDa) in the
culture media were markedly larger in size than those of
cellular forms in the cell lysate (38 and 34 kDa).
The MFG-E8, but not the C-domain deletion mutants,
is secreted as a constituent of high-molecular mass
complex and binds to phosphatidylserine
Molecular sizes of the MFG-E8-L, -S and its C -domain
deletion mutants secreted in the culture medium were
estimated by size exclusion ch romatography using a
Sephacryl S-300 column (Fig. 4). Both of the ELISA and

immunoblot analysis revealed that the wild-type proteins,
MFG-E8-L and -S, were eluted in the void volume fractions
(fraction numbers 16–18) much earlier than expected.
Therefore, the secreted MFG-E8 was found to behave as
high molecular mass complex(es). On the other hand, the
C-domain deletion mutants were eluted in fractions 26–28,
which corresponded t o t he elution volume for a 43-kDa
protein (ovalbumin). The molecular m asses estimated for
the C-domain deletion mutants by this size exclusion
chromatography agreed well with those by SDS/PAGE
(Fig. 3), indicating that the secreted DC1 and DC2 proteins
were monomeric. When the elution profiles of the two
C-domain deletion mutants were compared, the peak of
DC1 was obviously broader than that of DC2.
In some previous reports, the C2 domain of MFG-E8 as
well as that of blood clotting factors V and VIII has been
shown to be a binding-domain to PtdSer o r P tdSer-rich
Fig. 2. Cell surface localization of MFG-E 8 depending on the
C-domains. COS- 7 cells were transfected with plasmids containing
MFG-E8-L (A and B), MFG-E8-S (C an d D), DC1 (E and F) and DC2
(G and H) or empty plasmid (I and J). Transfectants were fixed and
stained with the antiserum specific for MFG-E8 followed by FITC-
labeled s econdary antibodies. The immunostaining was done with
permeabilization (B, D, F, H and J) or without (A, C, E, G and I). T he
cells were also stained with DAPI to visualize nuclei. Note that only
two wild-type MFG-E8s containing both of t wo C-domains (A and C)
were stained on cell surface, whereas intracellular MFG-E8 was
stained for all of the transfectants (B, D, F and H).
Fig. 1. A schematic representation o f M FG-E8 and its mutant proteins.
MFG-E8-L, a long form (a lactation mammary grand specific form) of

MFG-E8; MFG-E8-S, a short form (an ubiquitous form) of MFG-E8;
DC1, MF G-E8-L lacking C1 domain; DC2, MFG-E8- L lacking C2
domain. Signal sequence (SS), tandem EGF-like repeat (EGF1,
EGF2) and Pro/Thr-rich domain followed by two C-domains (C1, C 2)
are shown.
1212 K. Oshima et al. (Eur. J. Biochem. 269) Ó FEBS 2002
membrane [8,12,16]. Therefore, the PtdSer-binding a bility
of the s ecreted MFG-E8 and its C-domain deletion mutants
was examined by ELISA using polystylene microtiter plate
coated with PtdSer. MFG-E8-L and -S as well as DC1
showed the PtdSer-binding in a concentration-dependent
manner (Fig. 5). The DC2 protein, h owever, d id not bind to
the PtdSer-coated plates even at higher concentrations.
The high molecular mass complexes containing MFG-E8
are membrane-derived vesicles
The high molecular mass of secreted forms of MFG–E8
suggested certain interaction of an MFG-E8 molecule with
some other molecule(s) including MFG-E8 itself in a c ase o f
homophilic association. To determine w hether the secreted
MFG-E8 associates with proteins or other components
such as lipid, phospholipid and membrane vesicle, the
culture supernatant w as ultracentrifuged at 100 000 g in the
presence or absence of a nonionic detergent, Triton X-100,
and then both of the precipitate and supernatant were
subjected to Western blotting analysis for MFG-E8. As
shown i n Fig. 6, i n the absen ce o f the detergent, about a half
of the se creted MFG-E8 or more w as precipitated under
this centrifugation condition, whereas DC2 was not. The
sizes of MFG-E8-L, -S and DC2 bands seen in the
precipitates or sup ernatants were consistent with those of

the concentrated culture media Fig. 3, lanes 6–10. Interest-
Fig. 4. Size-exclusion chromatography of the secreted MFG-E8. Culture supernatants of MFG-E8-L (A), MFG-E8-S (B), DC1 (C) and DC2 (D)
transfectants w ere concentrated and applied t o a Sephacryl S -300 column. E ach fraction w as monitored by ELISA using the ant iserum s pecific f or
MFG-E8. The peak positions of blue dextran (V
0
) and ovalbumin ( 43 kDa) are indicated with arrowheads. Each fraction was analy zed by Western
blotting with the antiserum specific for MFG-E8 (lower panels).
Fig. 3. Western blot analyses for cell surface and s ecreted MFG-E8.
COS-7 cells were transfected with plasmids containing MFG-E8-L
(lanes 2, 7 and 12), MFG-E8-S (lanes 3, 8 and 13), DC1 (l anes 4, 9 and
14) a nd DC2 (lanes 5 , 10 an d 15) o r emp ty plasmid (lan es 1, 6 and 11).
Transfected CO S-7 cells were cultured in serum-free medium
(DMEM) for 24 h. T he cells were subjected to cell surface labeling with
sulfo-NHS-biotin and then lysed with the lysis buffer containing 1%
Triton X-100. Biotinylated proteins were precipitated with Streptavi-
din–Sepharose. The media were collected and concentrated by centri-
fugal filtration. Streptavidin-precipitates (lanes 1–5), the concentrated
media (lanes 6–10) and the total cell lysates (lanes 11–15) were analyzed
by SDS/PAGE followed by W estern blotting with the antiserum spe-
cific for MFG-E8. Note that only MFG-E8-L (lane 2) and MFG-E8-S
(lane 3 ) were biotin ylated, whereas all of the M FG-E8 and its mutants
were expressed a nd secreted (lanes 6–15). Position s o f molecular-mass
standards are indicated on the right.
Ó FEBS 2002 MFG-E8: a component of secreted membrane vesicles (Eur. J. Biochem. 269) 1213
ingly, MFG-E8 was no longe r precipitated when the
detergent was added to the culture supernatant prior to
the ultracentrifugation. We previously reported that
COMMA-1D cells endogenously expressed MFG-E8-S
and a small amount of MFG-E8-L mRNAs [9]. To clarify
whether MFG-E8 expressed in COMMA-1D cells was

secreted with membrane vesicles, the culture supernatant of
COMMA-1D cells was ultracentrifuged. By the Western
blotting analysis, two bands (66 and 51 kDa) of MFG-E8
were detected in the p recipitate, but not in the s upernatant
(Fig. 6), indicating that both of the MFG-E8 proteins
secreted by COMMA-ID cells were completely precipitated
under the ultracentrifugation condition used.
The precipitation by u ltracentrifugation at 100 000 g and
solubilization by Triton X-100 strongly suggested that the
secreted MFG-E8 was p resent in the culture medium as a
constituent of membrane vesicles, possibly in association
with membrane phospholipid. To confirm the assumption
that MFG-E8 was secreted as a component of membrane
vesicles, the culture supernatant containing secreted
MFG-E8 was subjected to the sucrose density-gradient
ultracentrifugation analysis. Figure 7 shows t ypical distri-
bution profiles with the density gradient for wild-type
MFG-E8 and the C-domain deletion mutants. Both of
MFG-E8-L and -S were detected in the fractions of lower
equilibrium-densities from 1.08 to 1.24. The two C-domain
deletion mutants, in contrast, we re not at all d etected in s uch
low-density fractions.
Thus, the MFG-E8 complex secreted in the culture
supernatant exhibited some characteristic properties, such
as higher sedimentation velocity, detergent s ensitivity and
lower specific gravity, which were just like those of the
microsome fraction of cell homogenates. To identify the
MFG-E8 complex as membrane vesicle, the MFG-E8
complex fraction re covered from the culture s upernatant by
the ultracentrifugation was observed under s canning elec-

tron microscop y. Some typical electron micrograms are
shown in Fig. 8, in which small vesicles with diameter in a
range of 100–200 nm and aggregations of the vesicles were
observed. The number of vesicles per microscopic field
(11.9 · 9.2 lm) was counted for randomly selected five
fields, and the average value for each transfectant is shown
in Fig. 8. The numbers of vesicles counted for MFG-E8-L
and - S were about 3–4 t imes that of DC2 or mock. The
counting for two independent transfectants gave similar
results.
DISCUSSION
MFG-E8 was originally identified as one of the major
MFGM g lycoproteins [1,15,27]. C loning of the MFG-E8
cDNA and structural analysis of t he predicted peptide
sequence has revealed that MFG-E8 lacks the transmem-
brane regions and is a peripheral membrane protein [1].
Many tissues besides the lactating mammary gland in some
mammals are reported to e xpress MFG-E8 [7–9]. Previous
reports have also shown that MFG-E8 is secreted into sera
of patients with breast tumor metastasis an d t he culture
supernatant of some cell lines [20,21,23], and that MFG-E8
purified from MFGM binds to avb5andavb3 i ntegrins and
promotes cell adhesion [7,11,12]. Therefore, MFG-E8 is
considered to contribute to cell–cell and/or cell–matrix
interactions in various tissues. Nevertheless, in spite o f the
cell adhesive ability, the localization of MFG-E8 in vivo
remains obscure, and it is not known even whether
MFG-E8 is a membrane bound protein or secretory. Here,
Fig. 5. In vitro PtdSer-binding of MFG-E8. Wells were coated with
PtdSer and blocked with 0 .05% gelatine. T hen various amounts of t he

culture supernatants of MFG-E 8-L (closed squares), MF G-E8-S
(open squares), DC1 (open triangles), DC2 (closed t riangles) and mock
(open circles) transfectants were added. After incubation, bidin g of
MFG-E8 to the PtdSer-coated plate was monitored with the antiserum
specific for MFG-E8.
Fig. 6. Detection of MFG-E8 in the membrane vesicle fraction. COS-7
cells w ere transfected with plasmids containing MFG-E8-L (lanes 1
and 2), M FG-E8-S (lanes 3– 6) and DC2 (lanes 7 a nd 8) and were also
cultured in a serum-free medium (DMEM) for 24 h. COMMA-1D
cells (lanes 9 and 10) were cultured in DMEM for 72 h. The culture
supernatants we re concentrated a n d sequentially c entrifuged at 1200 g
and 10 0 00 g to eliminate cells and debris. Then, the membrane vesi-
cles were pelle ted at 100 000 g. In some experiments, the media were
added 1 % Triton X -100 before the ultracentrifugatio n (lanes 5 and 6).
The resultant pellets (P) and sup ernatants (S) were analyzed by SDS/
PAGE followed by Western blotting with the ant iserum specific for
MFG-E8. Note that Triton X-100 treatment abrogated the recovery
of secreted MFG-E8 in the membrane v esicle fraction. Positions of
molecular-mass standards are indicated on the right.
1214 K. Oshima et al. (Eur. J. Biochem. 269) Ó FEBS 2002
we investigated the cellular localization of MFG-E8
expressed in COS-7 cells. The results of immunocyto-
chemistry (Fig. 2) and t he cell-surface b iotinylation study
(Fig. 3) clearly demonstrated that MFG-E8 was present
peripherally as small dots on the cell surface. On the
contrary, a transmembrane-type MFGM glycoprotein,
butyrophilin [27,28], expressed in COS-7 cells was detected
evenly the whole surface of the cells (Oshima, K.,
Fukushiro, A., Aoki, N., Kitajima, K. & Matsuda, T.,
unpublished data). Thus, MFG-E8 appeared to be unique

in such an uneven localization on plasma membrane. In
spite o f the cell-surface localization, MFG-E8-L and -S were
also found to be secreted to the culture supernatants
(Fig. 3 ). Secreted MFG-E8-L and - S were identified as 66
and 51 kDa by SDS/PAGE, respectively. However, t hey
were recovered only in the void volume fractions where
molecules with sizes higher than 150 kDa were eluted
(Fig. 4 ). Furthermore, the results of the ultracentrifugation
at 100 000 g (Fig. 6) and the sucrose density-gradient
ultracentrifugation (Fig. 7) suggested that secreted MFG-
E8 was associated with membrane vesicles. This was
strongly supported by solubilization of the MFG-E8
complex with Triton X-100 (Fig. 6). Recently, Thery et al.
reported that MFG-E8 is secreted from dendritic cell line,
D1, as a major c onstituent o f the exosome [20]. Indeed,
scanning e lectron microscopy revealed the existence of small
particles with a size ranging between 100 and 200 nm in the
culture supernatants from COS-7 cells (Fig. 8). Therefore, it
was suggested that COS-7 cells secreted exosome-like
membrane vesicles and that MFG-E8 was secreted as a
complex with the exosome-like membrane vesicles. It was
also observed that MFG-E8 expressed endogenously in
COMMA-1D cells was secreted and precipitated in the
membrane vesicle fraction (Fig. 6). Because COMMA-1D
cells was shown to e xpress both of MFG-E8-L and -S [9],
the 6 6 a nd 51 kDa band s s ecreted by COMMA-1D cells are
regarded a s t heir translational products, MFG-E8-L and - S,
respectively. The refore, this membrane vesicle association of
Fig. 7. Fractionation of secreted MFG-E8 by floatation on sucrose
density-gradient. COS-7 cells transfected with plasmids containing

MFG-E8-L, MFG-E8-S, DC1 and DC2 were cultured in serum-free
medium for 24 h. Culture supernatants were collected and concen-
trated by centrifugal filtration. After elimination of c ells and debris by
centrifugation, t he su pernatants were loaded on continuous sucrose
density-gradient (0.15–2.5
M
sucrose, resulting ranging 1.02–
1.32 gÆmL
)1
) followed by ultracentrifugation. The fractions were
recovered and analyzed by SDS/PAGE followed by Western blotting
with the antiserum specific for MFG-E8.
Fig. 8. Scanning electron micrographs of membrane particles derived from COS-7 cells transfected with MFG-E8. The precipitates at 100 000 g
obtained from the culture sup ernatants of MFG-E8-L (A), MFG-E8-S (B), DC2 (C) and mock (D) transfectants were analyzed by scanning
electron microsco py as describ ed in Experim ent al proced ures. Aggregates ob tain ed fro m MFG-E8-L (A) a nd m ock (D) transfectants were sho wn i n
the insets. Original magnification, 10 000 · ;Scalebar¼1lm. The number of the vesicles fro m each transfectant was cou nted for five different
microscopic fields and represented by an average ± S D (E).
Ó FEBS 2002 MFG-E8: a component of secreted membrane vesicles (Eur. J. Biochem. 269) 1215
MFG-E8 would not be due to artifacts resulted from the
overexpression in transformed heterologous cells. We also
tested whether butyrophilin expressed in COS-7 cells
associates with this exosome-like membrane vesicles. How-
ever, butyrophilin was r ecovered neither from the culture
supernatant nor the m embrane vesicle frac tion (Oshima, K.,
Aoki, N., Kitajima, K., & Matsuda, T., unpublished data).
Therefore, the membrane vesicles derived from C OS-7 cells
would accumulate MFG-E8 selectively.
The C2 domain of the blood clotting factor V and factor
VIII is essential for binding to PtdSer-rich membrane and
thus for procoagulant activity [17,29,30]. In agreement with

this, some investigators have shown that the C2 domain of
MFG-E8 is necessary for binding to PtdSer and the surface
of the MFGM and cells [12,14,16]. Therefore, MFG-E8 is
thought to bind to the membrane surface through the C2
domain. In the present study, however, not only DC2 but
also DC1 were shown to be monomeric (Fig. 4) and absent
on the cell surface (Figs 2 and 3 ) and in the m embrane
vesicle fraction (Fig. 7). These results i ndicate that both of
C1 and C2 domains of MFG-E8 are indispensable for the
association with the cell surface and the membrane vesicles.
In the in vitro assay system, on the other hand, DC1 lacking
the a bility to a ssociate with the cell surface a nd the exosome-
like membrane vesicles showed the PtdSer binding ability
(Fig. 5), indicating that only C2 domain was required and
enough for binding to PtdSer coated on the plate. This
binding by C2 domain alone might be due to a high density
of PtdSer on polystylene surface compared with cell
membrane. Thus, the C1 domain would also contribute as
a sub binding-domain to the MFG–E8 association with the
cell surface a nd the m embrane v esicles in vivo . The failure of
the DC1 and DC2 proteins to associate with the COS-7 cell
surface, form high molecular mass complexes and bind cell
membrane vesicles is not simply explained by a loss of
overall hydrophobicity, because the deletion of C1 domain
did not change the overall hydrophobicity. In fact, the DC1
protein had the PtdSer-binding ability probably t hrough the
remaining C2 domain regard ed as a ph ospholipid-binding
domain. Consequently, the membrane association of
MFG-E8-L and -S is supposed to be specific for the both
of C1 and C2 domain structures.

Some types of cells are known t o release lipid bilayer
vesicles b y unique mechanisms including apocrine, s hedding
and budding-off. The secretion of various membrane
vesicles into the extracellular space is a frequent phenom-
enon described i n normal and tumoral cells [31]. Hemato-
poietic cells, adhesive cells and tumor cells release two types
of membrane vesicles, exosomes and microvesicles, from
different mechanisms. In the present s tudy, scanning
electron microscopy showed that COS-7 cells sec reted small
particles with sizes ranging from 100 to 200 nm (Fig. 8).
This size range of the particles observed agrees well with this
exosomes a nd microvesicles. Exosomes have b een measured
40–100 nm i n d iameter. Exosomes originate f rom e ndocytic
multivesicular bodies (MVBs) and are released in an
exocytic manner [32]. Although functions of exosomes
remain largely to be resolved, they are thought to play
immunoregulatory a nd antitumoral r oles [20,32–34]. Micro-
vesicles have been measured from 100 nm to 1 lmin
diameter. Microvesicles originate from the cell surface
membrane and a re directly shedded i nto the extracellular
space [31,35–37]. Although they derive from the plasma
membrane, the shedded microvesicles have different lipid
and protein compositions [34,37–41]. Membrane shedding
is important for the membrane turn over and tumor
ganglioside metabolism [41]. Nevertheless, the processes of
exosome secretion and membrane shedding are scarcely
understood.
Two mechanisms for the secretion the exosome-like
membrane vesicles containing MFG-E8 have been hypoth-
esised from our present data and those of some other

investigators [20,31,32]. One h ypothesis is that MFG-E8 is
secreted as e xosomes in a n exocytic manner. COS-7 cells
expressed three size-variants ( 66, 56 and 51 kDa) for MFG-
E8-L on the cell surface but secreted only the 66-kDa form
(Fig. 3). Therefore, MFG-E8 may be secreted as exosomes
through a pathway differe nt from one transpo rting the cell
surface t ypes of MFG-E8. Exosomes s ecreted by B lympho-
cytes were recovered in the fractions corresponding to
densities of 1.0 8–1.22 gÆmL
)1
[42], similar t o the densities
where MFG-E8-L and -S were detected (1.08–1.24 gÆmL
)1
)
(Fig. 7). This also suggests an exosome-like secretion
mechanism. Another possible mechanism is membrane
shedding. The size of the small vesicles in COS-7 c ulture
medium resembles that of shedded microvesicles more
closely than that of exosomes previously reported [36,37, 40,
43], supporting the second mechanism. MFG-E8-L and -S
were detected as dot-like s taining, but butyrophilin was not.
It might be possible that MFG-E8 molecules are clustered
on the cell surface by binding to the particular regions or
molecules and th en released b y membrane s hedding to the
culture supernatant as a component of the membrane
vesicles. Approximately half of the MFG-E8, however,
remained in the high density fractions (Fig. 7), and
MFG-E8 was not completely precipitated by the ultracen-
trifugation (Fig. 6). These results imply that MFG-E8 was
also secreted as a complex with micelles. The exosome-like

membrane vesicles secreted by COS-7 cells would differ
from apoptotic vesicles, because DC2, which present in
cytoplasm, was precipitated at 10 000 g but not at
100 000 g (data not sho wn) [44].
We fo und that the DC2 and mock transfectants of COS-7
cells also secreted the exosome-like m embrane vesicles to the
culture supernatant. However, the vesicles and aggregates
were detected more in t he culture supernatants of MFG-E8-
L and -S transfectants than in those of the DC2 and mock
transfectants (Fig. 8). These results strongly suggest that
MFG-E8, membrane-associated t hrough the C2 domain,
plays a certain positive role i n the membrane secretio n by
some mammalian cells.
Milk lipids are synthesized in diffe rentiated mammary
epithelial cells and secreted from the apical side of the
cells as a droplet surrounded by plasma membrane
referred to as MFGM [45–47], in which considerable
amounts of MFG-E8 exist. The milk fat globules range in
size from under 0.2 to over 10 lmindiameter,and80%
or more of the total number of globules are below 1 lm.
Formation of the complex of butyrophilin, xanthine
oxidase a nd surfac e molecules of cytoplasmic lipid drop-
lets is speculated to b e essential for expulsion of milk fat
droplets [15,47]. The MFG-E8 secretion as membrane
vesicles observed in the present study suggests that
MFG-E8 expressed in the lactating mammary gland plays
specific roles in secretion of the milk lipid, especially of the
small lipid globules.
1216 K. Oshima et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ACKNOWLEDGEMENT

This research was supported in part b y Grants-in Aid for Scientific
Research from the Ministry of Edu cation, Sc ience, Sp orts and Culture
of Japan (to T. M., K. K., N. A. and K. O.).
REFERENCES
1. Stubbs, J.D., Lekutis, C., Singer, K .L., Bui, A ., Yuzuki, D.,
Srinivasan,U.&Parry,G.(1990)cDNAcloningofamouse
mammary e pithelial cell surface prot ein reveals the existen ce of
epidermal growth f actor-like domains linked to factor VIII-like
sequences. Proc. Natl Acad. Sci. USA 87, 8417–8421.
2. Aoki,N.,Kishi,M.,Taniguchi,Y.,Adachi,T.,Nakamura,R.&
Matsuda, T. (1995) Molecular cloning of glycoprotein antigens
MGP57/53 recognized by monoclonal antibodies raised against
bovine milk fat globule membrane. Biochim. Biophys. Acta 1245,
385–391.
3. Hvarregaard, J., Andersen, M.H., Berglund, L., Rasmussen, J.T.
& P etersen, T.E. (1996) Characterization of glycoprotein PAS-6/7
from membranes of bovine milk fat globules. Eur. J. Biochem. 240,
628–636.
4. Larocca, D., Peterson, J.A., Urrea, R., Kuniyoshi, J., Bistrain,
A.M. & Ceriani, R .L. (1991) A M
r
46 000 human milk fat globule
protein that is highly expressed in human breast tumors contains
factor VIII-like domains. Cancer Res. 51, 4994–4998.
5. Couto, J.R., Taylor, M.R., Godwin, S.G., Ceriani, R.L. &
Peterson, J.A. (1996) Cloning and sequence analysis of human
breast epithelial antigen BA46 reveals an RGD cell adhesion
sequence presented o n an epiderm al growth factor-like domain.
DNA Cell Biol. 15, 281–286.
6. Ogura, K., Nara,K., Watanabe, Y., K ohno, K., Tai, T. & Sanai, Y.

(1996) Cloning and expression of cDNA for O -acetylation of GD3
ganglioside. Biochem. Biophys. Res. Commun. 225, 932–938.
7. Andersen, M.H., Berglund, L., Rasmussen, J.T. & Petersen,
T.E. (1997 ) Bovine PAS-6/7 binds alpha v beta 5 integrins and
anionic p hospho lipids through two domains. Biochemistry 36,
5441–5446.
8. Aoki, N., Ishii, T., Ohira, S., Yamaguchi, Y., Negi, M., Adachi,
T., Nakamura, R. & Matsuda, T. (1997) S tage spe cific e xpression
of milk fat globule membrane glycoproteins in mouse mammary
gland: comparison of MFG-E8, b utyrophilin, and CD36 with
a major milk protein, b eta-casein. Biochim. Biophys. Acta 1334,
182–190.
9. Oshima, K., Aoki, N ., Ne gi, M ., Kishi, M., Kitajima, K. &
Matsuda, T. (1999) Lacta tion-dependent e xpression of an mRNA
splice variant with an exon for a multiply O-glycosylated domain
of mouse milk fat globule glycoprotein MFG-E8. Biochem. Bio-
phys. Res. Commun. 254, 522–528.
10. Hynes, R.O. (1992) Integrins: versatility, modulation, and signa-
ling in cell adhesion. Cell 69, 11–25.
11. Taylor, M.R., Couto, J.R., Scallan, C.D., Ceriani, R.L. &
Peterson, J.A. (1997) Lactadherin (formerly BA46), a m embrane-
associated glycoprotein expressed in human milk and breast
carcinomas, promotes Arg-Gly-Asp (RGD)-dependent cell
adhesion. DNA Cell Biol. 16, 861–869.
12. Andersen, M.H., Graversen, H., Fedosov, S .N., Petersen, T.E. &
Rasmussen, J.T. (2000) Functional analyses of two cellular
binding domains of bovine lactadherin. Biochemistry 39, 6200–
6206.
13. Basch, J .J., Farrell, H.M. & Greenberg, R. ( 1976) Identification of
the milk fat globule membrane proteins. I. Isolation and partial

characterization of glycoprotein B. Biochim. Biophys. Acta. 448 ,
589–598.
14. Peterson, J.A., Couto, J.R., Taylor, M.R. & Ceriani, R.L. (1995)
Selection of tumor-specific epitopes on target antigens for radio-
immunotherapyofbreastcancer.Cancer Res. 55, 5847–5851.
15. Mather, I.H. (2000) A review and proposed nomenclature for
major proteins of t he milk-fat globule membrane. J. D airy Sci. 83 ,
203–247.
16. Peterson, J.A., Patton, S. & Hamosh, M. (1 998) Gly coproteins of
the human milk fat globule in the protection of the breast-fed
infant against infections. Biol. Neonate 74, 143–162.
17. Pellequer, J.L., Gale, A.J., Griffin, J.H. & Getzoff, E.D. (1998)
Homology m odels of the C domains of blood c oagu lation factors
V and VIII: a proposed membrane binding mode for FV and
FVIII C2 domains. Blood Cells Mol. Dis. 24, 448–461.
18. Macedo-Ribeiro, S., Bode, W., Huber, R., Quinn-Allen , M.A.,
Kim, S.W., Ortel, T.L., Bourenkov, G.P., Bartunik, H.D., Stubbs,
M.T., Kane, W.H. & Fuentes-Prior, P. (1999) Crystal structures of
the membrane-binding C2 domain of human coagulation factor
V. Nature 402, 434–439.
19. Pratt, K.P., Shen, B.W., Takeshima, K., Davie, E.W., Fujikawa,
K. & Stoddard, B.L. ( 1999) Structure of the C2 domain of human
factor VIII at 1.5 A
˚
resolution. Nature 402, 439–442.
20. Thery, C., Regnault, A., Garin, J., Wolfers, J., Zitvogel, L.,
Ricciardi-Castagnoli, P., Raposo, G. & Amigorena, S. (1999)
Molecular characterization of dendritic cell-derived exosomes.
Selective accumulation of the heat shock protein hsc73. J. Cell.
Biol. 147, 599–610.

21. Goldberg, G.S., B echberger, J.F., Tajima, Y., Merritt, M., Omori,
Y., Gawinowicz, M.A., Narayanan, R., Tan, Y., Sanai, Y.,
Yamasaki, H., Naus, C.C., Tsuda, H. & Nicholson, B.J. (2000)
Connexin43 suppresses MFG-E8 while inducing contact growth
inhibition of glioma cells. Cancer Res. 60, 6018–6026.
22. Kanai, Y., Kanai-A zuma, M., T ajima, Y., Birk, O.S., Hayashi, Y.
& Sanai, Y. (2000) Identification of a s tromal cell type char-
acterized by the secretion of a soluble integrin-binding protein,
MFG-E8, in mouse e arly go nadogenesis. Mech. Dev. 96, 223–227.
23. Salinas, F.A., Wee, K.H. & C eriani, R.L. (1987) Significance of
breast carcinoma-associated antigens as a monitor of tumor bur-
den: characterization by monoclonal antibodies. Cancer Res. 47,
907–913.
24. Chen, C. & Okayama, H. (1987) High-efficiency transformation of
mammalian cells by plasmid DNA. Mol . Cell. Biol. 7, 274 5–2752.
25. Aoki, N., Kuroda, H., Urabe, M., Taniguchi, Y., Adachi, T.,
Nakamura, R. & Matsuda, T. (1994) Production and char-
acterization of monoclonal an tibodies directed against bovine
milk fat globule membrane (MFGM). Biochim. Biophys. Acta
1199, 87–95.
26. Martinez-Lorenzo, M.J., Anel, A., Gamen, S., Monle, N.I.,
Lasierra, P., Larrad, L., Pineiro, A., Alava, M.A. & Naval, J.
(1999) Activated human T cells release bioactive Fas ligand and
APO2 ligand in microvesicles. J. Immunol. 163, 1274–1281.
27. Mather, I.H., Tamplin, C.B. & Irving, M.G. (1980) Separa tion of
the proteins of b ovine milk-fat- globule membrane b y electro-
focusing with retention of enzymatic and immunological activity.
Eur. J. Biochem. 110, 327–336.
28. Mather, I.H. & Jack, L.J. (1993) A review of the molecular and
cellular biology of butyrophilin, t he major protein of bovine milk

fat globule membrane. J. Dairy Sci. 76, 3832–3850.
29. Isaacs, B.S., Husten, E.J., Esmon, C.T. & Johnson, A.E. (1986)
A domain of membrane-bound blood coagulation factor Va is
located far from the phospholipid surface. A fluorescence energy
transfer measurement. Biochemistry 25, 4958–4969.
30. Kalafatis, M., Jenny, R.J. & Mann, K.G. (1990) Identification and
characterization of a phospholipid-binding site of bovine factor
Va. J. Biol. Chem. 265, 21580–21589.
31. Dainiak, N. (1991) Surface membrane-associated regulation of cell
assembly, differentiation, and growth. Blood 78, 264–276.
32. Denzer, K., Kleijmeer, M.J., Heijnen, H.F., Stoorvogel, W. &
Geuze, H.J. (2000) E xosome: from internal vesicle of the multi-
vesicular body to intercellular signaling device. J. Cell. Sci. 113
Part 19, 3365–3374.
Ó FEBS 2002 MFG-E8: a component of secreted membrane vesicles (Eur. J. Biochem. 269) 1217
33. Zitvogel, L., Regnault, A., Lozier, A., Wolfers, J., F lament, C.,
Tenza, D., R icciardi-Castagnoli, P., R aposo, G. & Amigorena, S.
(1998) Eradication of established murine tumors using a novel cell-
free vaccine: de ndritic cell-derived exosomes. Nat. Med. 4,594–
600.
34. Wolfers, J., Lozier, A., R aposo, G., Regnault, A., Thery, C.,
Masurier, C., Flament, C., Pouzieux, S ., Faure, F., Tursz, T.,
Angevin, E., Amigorena, S. & Zitvogel, L. (2001) Tumor- derived
exosomes are a source of shared tumor rejection antigens for CTL
cross-priming. Nat. Med. 7, 297–303.
35. Dolo, V., Ginestra, A., Cassara, D., Violini, S., Lucania, G .,
Torrisi, M.R., Nagase, H., Canevari, S., Pavan, A. & Vittorelli,
M.L. (1998) Selective localization of matrix metalloproteinase 9,
beta1 integrins, and human lymphocyte antigen class I molecules
on membrane vesicles shed by 8701-BC breast carcinoma cells.

Cancer Res. 58, 4468–4474.
36. Heijnen, H.F., Schiel, A.E., Fijnheer, R., Geuze, H.J. & Sixma,
J.J. (1999) Activated platelets release two types o f membrane
vesicles: microvesicles by surface shedding and exosomes derived
from exocytosis of multivesicular bodies and alpha-granules.
Blood 94, 3791–3799.
37. Dolo, V., Li, R., Dillinger, M., Flati, S., Manela, J., Taylor, B.J.,
Pavan, A. & Ladisch, S. (2000) Enrichment and localization of
ganglioside G (D3) and caveolin-1 in shed tumor cell membrane
vesicles. Biochim. Biophys. Acta. 1486, 265–274.
38. Van Blitterswijk, W.J., De Veer, G., Krol, J.H. & Emmelot, P.
(1982) Comparative lipid analysis of purified plasma membranes
and shed extracellular membrane vesicles from normal murine
thymocytes and leukemic GRSL cells. Biochim. Biophys. Acta.
688, 495–504.
39. Lerner, M.P., Lucid, S.W., Wen, G.J. & Nordquist, R.E. (1983)
Selected area membrane shedding by tum or cells. Cancer Lett. 20,
125–130.
40. Ginestra, A., Monea, S., Seghezzi, G., Dolo, V., Nagase, H .,
Mignatti, P. & Vittorelli, M.L. ( 1997) Urokinase p lasminogen
activator and gelatinases are associated with membrane vesicles
shed by human HT1080 fibrosarcoma cells. J. Biol. C hem. 272,
17216–17222.
41. Kong, Y., Li, R. & Ladisch, S. (1998) Natural forms of shed tumor
gangliosides. Biochim. Biophys. Acta 1394, 43–56.
42. Raposo, G., Nijman, H.W., Stoorvogel, W., Liejendekker, R .,
Harding, C.V., Melief, C.J. & Geuze, H.J. (1996) B lymphocytes
secrete antigen-presenting vesicles. J. Exp. Med. 183, 1161–1172.
43. Clayton, A., Court, J., N avabi, H., Adams, M., Mason, M.D.,
Hobot, J.A., Newman, G.R. & Jasani, B. (2 001) Analy sis of

antigen presenting cell derived exosomes, based on immuno-
magnetic isolation and flow cytometry. J Immunol. Methods 247,
163–174.
44. Thery, C., Boussac, M ., V eron, P., Ricciardi-Castagnoli, P., Rap-
oso, G., Garin, J. & Amigorena, S. (2001) Proteomic analysis of
dendritic cell-derived exosomes: a secreted subcellular compart-
ment d ist inct from apoptotic vesicles. J Immunol. 166, 7309–7818.
45. Mather, I.H. (1987) The Mammary Grand, Development, Regula-
tion, and Function (Neville, M.C. & Daniel, C.W., eds), pp. 217–
267. Plenum Publishing Corp, New York.
46. Deyrup-Olsen, I. & Luchtel, D.L. (1998) Secretion of mucous
granules and other membran e-bound structures: a look beyond
exocytosis. Int. Rev. Cytol. 183, 95–141.
47. Mather, I .H. & Keenan, T.W. ( 1998) Origin a nd secretion o f milk
lipids. J. Mamm. Gland Biol. Neoplasia 3, 259–273.
1218 K. Oshima et al. (Eur. J. Biochem. 269) Ó FEBS 2002