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: tmatsuda@agr.nagoya-u.ac.jp 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. 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Oshima et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Secretion of a peripheral membrane protein, MFG-E8, as a complex with membrane vesicles A possible role in membrane secretion Kenji Oshima 1 , Naohito. 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