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

Eur. J. Biochem. 269, 1209–1218 (2002) (cid:211) FEBS 2002

Secretion of a peripheral membrane protein, MFG-E8, as a complex with membrane vesicles A possible role in membrane secretion

Kenji Oshima1, Naohito Aoki1, Takeo Kato2, Ken Kitajima1 and Tsukasa Matsuda1 1Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan; 2Food Research Institute, Aichi Prefectural Government, Nagoya, Japan

characterized to have a high-molecular mass, low density and higher sedimentation velocity and to be detergent- sensitive. Not only such a exogenously expressed MFG-E8 but also that endogenously expressed in a mammary epi- thelial cell line, COMMA-1D, was secreted as the membrane vesicle-like complex. Scanning electron microscopic analyses revealed that MFG-E8 was secreted into the culture medium in association with small membrane vesicles with 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 of MFG-E8 in the membrane vesicle secretion, such as budding or shedding of plasma membrane (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) is a peripheral 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-like domains, a mucin-like domain and two-repeated discoidin- like domains (C-domains), and contains an integrin-binding motif (RGD sequence) in the EGF-like domain. To clarify the role of 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 staining of intracellular 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 for the membrane association. During the course of these studies for domain functions, MFG-E8, but not C-domain deletion mutants, was shown to be secreted as membrane vesicle complexes. By size-exclusion chromato- graphy and ultracentrifugation analyses, the complexes were

bovine [7–9]. The mouse and bovine MFG-E8 proteins expressed in mammary gland were shown to 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].

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 C2 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

especially phosphatidylserine

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 avb3 and avb5 integrins [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 (PtdSer) phospholipids, [7,12,16]. This PtdSer-binding of MFG-E8 has been reported to depend only on the second C-domain (C2-domain), but not the first C-domain (C1-domain), in the same manner as that of 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, Japan. Fax: + 81 52 789 4128, Tel.: + 81 52 789 4129, E-mail: tmatsuda@agr.nagoya-u.ac.jp Abbreviations: MFGM, milk fat globule membrane; DMEM, 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)

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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 density of 2.5 · 105 cells per 60-mm dish, and 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% CO2 and 97% air for 18 h, the transfected cells were washed with NaCl/Pi and cultured under humidified 5% CO2 and 95% air.

Immunofluorescence staining

Recently, MFG-E8 was detected as the major component of the secretory membrane vesicle (exosome) secreted by a murine dendritic cell line (D1) [20]. Furthermore, a glioma cell line (C6) has also 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 uncharacterized 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.

E X P E R I M E N T A L P R O C E D U R E S

Cell culture

COS-7 cells were cultured on cover glasses and transfected with MFG-E8s expression plasmids as described above. After being cultured in DMEM containing 10% fetal bovine serum for 24 h, cells were washed three times with NaCl/Pi and fixed with 3% paraformaldehyde in NaCl/Pi for 8 min for extracellular staining or with methanol chilled at )20 (cid:176)C for 5 min for intracellular staining. After blocking with NaCl/Pi containing 2% BSA (blocking solution) for 30 min, the specimens were incubated for 60 min with the rabbit antiserum 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) Ig (ICN/Cappel). Samples were then incubated for 15 min with 4¢,6-diamidine-2-phenylindole-dihydrochlo- ride (DAPI) (Roche Molecular Biochemicals) (1 lgÆmL)1 NaCl/Pi) and washed three times with NaCl/Pi. Images were acquired by using a fluorescence microscope (Olympus).

A mouse mammary epithelial cell line, COMMA-1D, and a monkey kidney cell line, COS-7 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) containing 10% heat-inactivated fetal bovine serum, penicillin at 100 UÆmL)1, streptomycin at 100 lgÆmL)1 at 37 (cid:176)C under humidified 5% CO2 and 95% air.

SDS/PAGE and Western blotting

Construction of expression plasmids and gene transfection

The transfected cells were cultured in serum-free DMEM for 24 h and then lysed with (cid:212)lysis buffer(cid:213) containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 5 mM EDTA, 1 mM 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 Mr 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

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 and downstream of the C1 domain were amplified by PCR using the MFG-E8-L expression plasmid as a template with primer sets containing an XbaI site as follows: 5¢-ATGCAGGTCTCCCGTGTGC-3¢, 5¢-ATTCTAGAG GCTAGGTTGTTGGAAAG-3¢, 5¢-ATTCTAGAGGAT 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 arginine, which were translated from the XbaI- site sequence TCTAGA. The cDNA lacking the C1 domain was then amplified by PCR from the cloned plasmid with primers containing an EcoRI site at the 5¢ end (5¢-TAGAATTCCACCATGCAGGTCTCCCGT-3¢) and an EcoRV site at the 3¢ end (5¢-CAGATATCTTAACAGC CCAGCAGCTC-3¢). The cDNA lacking the C2 domain (amino acids 307–463) was created by PCR directly from the MFG-E8-L expression plasmid with primers containing an EcoRI site at the 5¢ end as same above and an EcoRV

COS-7 cells were plated onto six-well polystyrene plates at a density of 1 · 105 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/Pi and incubated at 4 (cid:176)C for 30 min in the presence of 0.5 mgÆmL)1 Sulfo-N-hydroxysulfosuccinimide-

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in

(Sigma)

Biotin (Pierce). After nonreacted biotin was quenched with serum-free DMEM at 4 (cid:176)C for 5 min, cells were washed three times with NaCl/Pi and then lysed with the lysis buffer. Streptavidin–Sepharose (Amersham Pharmacia Biotech) was added to the cell lysate and incubated overnight at 4 (cid:176)C. Proteins bound to the Sepharose were precipitated by centrifugation and washed extensively with a buffer containing 50 mM Hepes (pH 7.5), 150 mM 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

L-a-phosphatidyl-L-serine methanol (10 lgÆmL)1) was added to a micro well plate (Nunc) (30 lLÆwell)1) followed by drying at 37 (cid:176)C. The plate was washed three times between all subsequent steps with NaCl/ Tris containing 0.05% Tween-20. The plate was blocked with 200 lL of NaCl/Tris containing 0.05% (w/v) gelatine (blocking buffer). Culture supernatants of the transfected 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 by incubation at 4 (cid:176)C overnight. The plate was then incubated with anti-(GST– MFG-E8) serum and peroxidase-labeled goat anti-(rabbit IgG) Ig as the secondary antibody, and peroxidase activity was measured.

Scanning electron microscopy

a Sephacryl S-300

Culture supernatants from the transfected COS-7 cells (8 · 106 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 column (0.9 · 60 cm), equilibrated with NaCl/Pi. 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 anti- (GST–MFG-E8) serum and peroxidase-conjugated goat anti-(rabbit IgG) Ig as described previously [25].

Samples for scanning electron microscopy analysis were prepared essentially as previously described for microvesi- cles [26]. Culture supernatants of the transfected COS-7 cells (2 · 106 cells) cultured in serum-free DMEM for 48 h were centrifuged at 10 000 g for 30 min to eliminate cells and debris. The supernatants were then centrifuged at 200 000 g for 1.5 h at 4 (cid:176)C. The pellets were resuspended in 100 lL of NaCl/Tris with 0.01% sodium azide and 10 lgÆmL)1 leupeptin. The suspended samples were placed onto micro- scope glass slides, previously treated with poly L-lysine (Sigma) for 30 min, and then fixed with 1% OsO4 for 2 h. The samples were dehydrated in a series of ethanol (50–100%), critical-point dried in a CO2 system. After being platinum/palladium-coated in a spattering devise, the speci- mens were observed with a scanning electron microscope (JSM-820, Japan Electron Optics Laboratory).

R E S U L T S

Cellular and extracellular distribution of MFG-E8 and its domain deletion mutants expressed in COS-7 cells

Ultracentrifugation Culture supernatants from COMMA-1D cells (8 · 106 cells) and the transfected COS-7 cells (4 · 106 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 by the 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 (cid:176)C. The resulting supernatants were recovered, while the pellets were resuspended in 100 lL of NaCl/Pi 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 · 106 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 mM Tris/HCl (pH 7.5) containing 150 mM NaCl and 5 mM 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 (cid:176)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

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 shown in Fig. 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 permeable 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, expressed in COS-7 cells, the two localized on the cell surface and both of C-domains were indispensable for such a cell surface localization of MFG-E8. A possibility that cytosolic MFG-E8 was stained under the nonpermeable condition could be excluded because DC1 and DC2 were not detected under the same condition.

The ELISA for MFG-E8 binding to solid-phase phospho- [7]. lipid was performed as described previously

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As the expression and localization 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 biotinylation, confirming their existence on the cell surface. In contrast, only weak or almost no bands were detected for the C-domain deletion mutants, indicating that they did not retain on the cell surface. When the culture supernatants were analyzed, considerable amounts of the C-domain deletion mutants were found to be secreted into the culture medium. Furthermore, MFG-E8-L and -S were detected in culture supernatant, indicating that they were not only plasma-membrane-associated but also secreted. While three size-variants (66, 56 and 51 kDa) for MFG-E8-L were detected in the total cell lysate and cell-surface biotinylated proteins, secreted 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 or cellular 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

Fig. 1. A schematic representation of MFG-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, MFG-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, C2) are shown.

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 or PtdSer-rich

Molecular sizes of the MFG-E8-L, -S and its C-domain deletion mutants secreted in the culture medium were estimated by size exclusion chromatography 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 to the elution volume for a 43-kDa protein (ovalbumin). The molecular masses estimated for

Fig. 2. Cell surface localization of MFG-E8 depending on the C-domains. COS-7 cells were transfected with plasmids containing MFG-E8-L (A and B), MFG-E8-S (C and 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 secondary antibodies. The immunostaining was done with permeabilization (B, D, F, H and J) or without (A, C, E, G and I). The cells were also stained with DAPI to visualize nuclei. Note that only two wild-type MFG-E8s containing both of two 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).

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membrane [8,12,16]. Therefore, the PtdSer-binding ability of the secreted 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, however, did 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 case of homophilic association. To determine whether the secreted MFG-E8 associates with proteins or other components such as lipid, phospholipid and membrane vesicle, the culture supernatant was 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 in Fig. 6, in the absence of the detergent, about a half of the secreted MFG-E8 or more was precipitated under this centrifugation condition, whereas DC2 was not. The sizes of MFG-E8-L, -S and DC2 bands seen in the precipitates or supernatants were consistent with those of the concentrated culture media Fig. 3, lanes 6–10. Interest-

Fig. 3. Western blot analyses for cell surface and secreted 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 (lanes 4, 9 and 14) and DC2 (lanes 5, 10 and 15) or empty plasmid (lanes 1, 6 and 11). Transfected COS-7 cells were cultured in serum-free medium (DMEM) for 24 h. The 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 Western blotting with the antiserum spe- cific for MFG-E8. Note that only MFG-E8-L (lane 2) and MFG-E8-S (lane 3) were biotinylated, whereas all of the MFG-E8 and its mutants were expressed and secreted (lanes 6–15). Positions of molecular-mass standards are indicated on the right.

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 were concentrated and applied to a Sephacryl S-300 column. Each fraction was monitored by ELISA using the antiserum specific for MFG-E8. The peak positions of blue dextran (V0) and ovalbumin (43 kDa) are indicated with arrowheads. Each fraction was analyzed by Western blotting with the antiserum specific for MFG-E8 (lower panels).

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Fig. 5. In vitro PtdSer-binding of MFG-E8. Wells were coated with PtdSer and blocked with 0.05% gelatine. Then various amounts of the culture supernatants of MFG-E8-L (closed squares), MFG-E8-S (open squares), DC1 (open triangles), DC2 (closed triangles) and mock (open circles) transfectants were added. After incubation, biding of MFG-E8 to the PtdSer-coated plate was monitored with the antiserum specific for MFG-E8.

ingly, MFG-E8 was no longer 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 precipitate, but not in the supernatant (Fig. 6), indicating that both of the MFG-E8 proteins secreted by COMMA-ID cells were completely precipitated under the ultracentrifugation condition used.

complex fraction recovered from the culture supernatant by the ultracentrifugation was observed under scanning elec- tron microscopy. 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 times that of DC2 or mock. The counting for two independent transfectants gave similar results.

Fig. 6. Detection of MFG-E8 in the membrane vesicle fraction. COS-7 cells were transfected with plasmids containing MFG-E8-L (lanes 1 and 2), MFG-E8-S (lanes 3–6) and DC2 (lanes 7 and 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 were concentrated and sequentially centrifuged at 1200 g and 10 000 g to eliminate cells and debris. Then, the membrane vesi- cles were pelleted at 100 000 g. In some experiments, the media were added 1% Triton X-100 before the ultracentrifugation (lanes 5 and 6). The resultant pellets (P) and supernatants (S) were analyzed by SDS/ PAGE followed by Western blotting with the antiserum specific for MFG-E8. Note that Triton X-100 treatment abrogated the recovery of secreted MFG-E8 in the membrane vesicle fraction. Positions of molecular-mass standards are indicated on the right.

D I S C U S S I O N

The precipitation by ultracentrifugation at 100 000 g and solubilization by Triton X-100 strongly suggested that the secreted MFG-E8 was present 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 the culture supernatant containing secreted vesicles, MFG-E8 was subjected to the sucrose density-gradient ultracentrifugation analysis. Figure 7 shows typical 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, were not at all detected in such low-density fractions.

Thus, the MFG-E8 complex secreted in the culture supernatant exhibited some characteristic properties, such as higher sedimentation velocity, detergent sensitivity 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

MFG-E8 was originally identified as one of the major MFGM glycoproteins [1,15,27]. Cloning of the MFG-E8 cDNA and structural analysis of the 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 express MFG-E8 [7–9]. Previous reports have also shown that MFG-E8 is secreted into sera of patients with breast tumor metastasis and the culture supernatant of some cell lines [20,21,23], and that MFG-E8 purified from MFGM binds to avb5 and avb3 integrins 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 of 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,

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resulting ranging (0.15–2.5 M sucrose,

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 of 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, they 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 constituent of the exosome [20]. Indeed, scanning electron 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 express both of MFG-E8-L and -S [9], the 66 and 51 kDa bands secreted by COMMA-1D cells are regarded as their translational products, MFG-E8-L and -S, respectively. Therefore, this membrane vesicle association of

we investigated the cellular localization of MFG-E8 expressed in COS-7 cells. The results of immunocyto- chemistry (Fig. 2) and the cell-surface biotinylation study (Fig. 3) clearly demonstrated that MFG-E8 was present

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 cells and debris by centrifugation, the supernatants were loaded on continuous sucrose 1.02– density-gradient 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 supernatants of MFG-E8-L (A), MFG-E8-S (B), DC2 (C) and mock (D) transfectants were analyzed by scanning electron microscopy as described in Experimental procedures. Aggregates obtained from MFG-E8-L (A) and mock (D) transfectants were shown in the insets. Original magnification, 10 000 · ; Scale bar (cid:136) 1 lm. The number of the vesicles from each transfectant was counted for five different microscopic fields and represented by an average (cid:139) SD (E).

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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.

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 recovered neither from the culture supernatant nor the membrane vesicle fraction (Oshima, K., Aoki, N., Kitajima, K., & Matsuda, T., unpublished data). Therefore, the membrane vesicles derived from COS-7 cells would accumulate MFG-E8 selectively.

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 hypothesis is that MFG-E8 is secreted as exosomes in an 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 different from one transporting the cell surface types of MFG-E8. Exosomes secreted by B lympho- cytes were recovered in the fractions corresponding to densities of 1.08–1.22 gÆmL)1 [42], similar to 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 culture 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 staining, 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 then released by membrane shedding 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 shown) [44].

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 membrane vesicle fraction (Fig. 7). These results indicate 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 ability to associate with the cell surface and 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 and the membrane vesicles 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 through the remaining C2 domain regarded as a phospholipid-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.

We found that the DC2 and mock transfectants of COS-7 cells also secreted the exosome-like membrane vesicles to the culture supernatant. However, the vesicles and aggregates were detected more in the 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 through the C2 domain, plays a certain positive role in the membrane secretion by some mammalian cells.

Some types of cells are known to release lipid bilayer vesicles by unique mechanisms including apocrine, shedding and budding-off. The secretion of various membrane vesicles into the extracellular space is a frequent phenom- enon described in 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 study, scanning electron microscopy showed that COS-7 cells secreted small particles with sizes ranging from 100 to 200 nm (Fig. 8). This size range of the particles observed agrees well with this exosomes and microvesicles. Exosomes have been measured 40–100 nm in diameter. Exosomes originate from endocytic 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 and antitumoral roles [20,32–34]. Micro- vesicles have been measured from 100 nm to 1 lm in diameter. Microvesicles originate from the cell surface membrane and are directly shedded into the extracellular space [31,35–37]. Although they derive from the plasma

Milk lipids are synthesized in differentiated 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 lm in diameter, and 80% or more of the total number of globules are below 1 lm. Formation of the complex of butyrophilin, xanthine oxidase and surface molecules of cytoplasmic lipid drop- lets is speculated to be 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.

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A C K N O W L E D G E M E N T

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This research was supported in part by Grants-in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to T. M., K. K., N. A. and K. O.). 16. Peterson, J.A., Patton, S. & Hamosh, M. (1998) Glycoproteins of the human milk fat globule in the protection of the breast-fed infant against infections. Biol. Neonate 74, 143–162.

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