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

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)

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

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

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

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

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

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

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

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-

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

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

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

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
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