Location of glycoproteins on milk fat globule membrane by scanning and transmission electron microscopy, using lectin-labelled gold granules

LOCATION

OF GLYCOPROTEINS

MEMBRANE

BY SCANNING

ELECTRON

Research

Department,

AND TRANSMISSION

MICROSCOPY,

LECTIN-LABELLED M. HORISBERGER,

ON MILK FAT GLOBULE USING

GOLD GRANULES

J. ROSSET and M. VONLANTHEN

Nestlk Products Technical Assistance

Co. Ltd.,

CH-1814 La Tour de Peilz, Switzerland

SUMMARY Membrane glycoproteins of bovine and human milk fat globules (MFG) were located by scanning electron microscopy using lectin-labelled gold granules (50 nm diameter) as specific markers. Receptors for wheat germ agglutinin (WGA) and soybean lectin (SBA) were localized in clusters over the whole MFG surface. When MFG were treated with neuraminidase, the density of marking with SBA increased. Marking of MFG with Concanavalin A (ConA) was weak. No marking was obtained with lectins specific for L-fucose, n-galactose and a-o-galactose. When thin sections of MFG marked with WGA (18 nm diameter gold granules) were examined by transmission electron microscopy, the membrane was uniformly marked. Using markers of different sizes (5 and 18 nm diam.) prefixed milk fat globule membranes (MFGM) were simultaneously marked with WGA and SBA. The lectin receptors appeared to belong to different glycoproteins which were clustered. Thin sections of this material showed that the receptors were located on one side of the membrane. No difference was observed between bovine MFG and human MFG from donors having blood group 0 and A. All results indicated that MFGM is a true biological membrane.

The composition and structure of milk fat globule (MFG) and milk fat globule membrane (MFGM) have been recently reviewed by Anderson & Cawston, and by Patton & Keenan [l, 21. MFGM is composed of neutral and polar lipids, trace elements, enzymes, proteins and glycoproteins. It is generally accepted that during secretion the globule is enveloped by an outer unit membrane (originating directly from the apical membrane of the lactating cell and possibly from Golgi vesicle membranes) and by an inner interfacial layer adjacent to the core lipid. In between is an intermediate dense layer partly or wholly derived from the cytoplasm of the lactating

cell [3-51. The outside of the lactating cell membrane becomes the exposed surface of MFG which is thought to be a true biological membrane. Up to nine periodate-Schiff positive glycoprotein bands have been detected in electrophoretic patterns of MFGM [2] and one glycoprotein has been partially characterized [6]. Lectins have been extensively used for probing glycoproteins of biological membranes. Different lectins have different specificities: Concanavalin A (ConA) binds to a-D-mannose or cY-D-glUCOSe, soybean agglutinin (SBA) to D-galactose and Nacetyl-D-galactosamine, wheat germ aggluExp

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Horisberger, Rosset and Vonlanthen

tinin (WGA) to N-acetyl-D-ghXosamine containing oligosaccharides, peanut lectin to D-galactose, anti-H lectin (Ufex europaeus) to L-fucose and di-Wacetyl-chitobiose [7], and lectin from Bandeiraea simplicifolia to a-D-galactose [8]. Keenan et al. [9] concluded that ConA-binding sites were disposed externally on MFG since isolated membranes and intact fat globules reacted both to the same extent with tritiated ConA. The same asymmetry was found for sialic acid [lo]. In this study, glycoproteins were located on bovine and human MFG by scanning electron microscopy (SEM) and on bovine MFGM by transmission electron microscopy (TEM) using lectins as markers. The markers were prepared by a method recently developed using gold granules of sizes suitable for TEM [ 11, 121 and SEM

[131. MATERIALS

AND METHODS

Materials Milk was obtained from Holstein Friesian cows and human milk from a hospital. Concanavalin A (ConA) was from Miles Laboratories, soybean agglutinin (SBA) from Pharmacia Fine Chemicals, wheat germ agglutinin (WGA) from 1’Industrie Biologique FranCaise (Genevilliers, France), peanut lectin and anti-H lectin (Ufex europaeus) from P.L. Biochemicals (Milwaukee), chloroauric acid (HAuCl, aq., purum 50% Au) was from Fluka (Buchs, Switzerland), polyethylene glycol (Carbowax 20 M) from Union Carbide Chemicals Co. (New York), and neuraminidase (500 U/ml, B grade, Vibrio chlorerue) from Calbiochem. N-Acetyl chitopentaose was prepared by the method of Rupley [ 141and the lectin from Bandeiroea simplicifolk by affinity chromatography on a polyacrylamide gel containing guar gum [ 1.51.

Labelling of gold granules The smallest size gold granules (AuI) were obtained by reducing a solution of HAuCl, with white phosphorus [ 1] and larger size granules (Au II and III) were prepared by boiling 0.01% HAuCI, (100 ml) with 1% sodium citrate (2 and 1 ml, respectively) [16, 171.The mean diameter measured on 100granules was 5,26 and 50 nm for Au I, II and III. The optimum amount of lectin necessary to label Au I was determined as described elsewhere 111, 12, Exp CellRes 109 (1977)

171using NaCl as the flocculating agent. For Au II and III, the procedure was modified: Gold granules (5 ml) were mixed with increasing amounts of lectin diluted in 0.005 M NaCl. DH 7.0 (0.5 ml). After 1 h at 25°C the addition of an&ufIicient amount of protein caused aeglutination of the colloid as seen bv a decrease in the a&orbance at the maximum of absorption of the colloid (530-550 nm). Stable gold markers could not be obtained with WGA due to its small molecular weight. WGA was therefore cross-linked with glutaraldehyde to bovine serum albumin as described e&lier [12]. Gold granules (100 ml) were then labelled with a 10% excess of lectin accordine to oublished orocedures [12, 13, 17, 181. The gra&les were centhfuged at 63 000 e (60 min. AuI). 28000 e (60 min. Au II; 20 min A;IiI) and the pellets were-suspended to an outical densitv of 10 at the maximum of absorption of tGe markers (520-540 nm) in 0.05 M Tris, 0.15 h;r NaCl, pH 7.4, containing 0.5 mg sodium azide and Carbowax 20 M (buffer A) or in buffer A made 0.001 M in MnCl, and CaCl, in the case of ConA gold markers (buffer B). The markers were kept at 4°C.

Isolation of milk fat globules Milk (10 ml) was centrifuged at 2000 g for 5 min at 25°C and the cream was washed twice with buffer A and fixed for 15 min in buffer A containing 0.25% glutaraldehyde. MFG were washed twice and suspended in buffer A at a concentration of IO9MFG/ml which was estimated with a hemacytometer.

Isolation of milk fat globule membranes MFGM were prepared from cow’s milk by the procedure of Keenan et al. [19]. MFGM were isolated in buffer B and fixed for 15 min at 25°C in buffer B containing 0.25% glutaraldehyde. MFGM were centri. fuged and resuspended in buffer A (AesOnm13.0).

Incubation of milk fat globules with neuraminidase Human MFG were dispersed in phosphate-buffered saline, pH 7.4 (3.6 ml, 5X10* MFG/ml) and were incubated for 1 h at 37°C with 0.4 ml neuraminidase. They were centrifuged and washed twice with buffer A, fixed for 15 min at 25°C in buffer A containing 0.25 % glutaraldehyde. After two washings, they were suspended in buffer A to a final concentration of lo9 MFG/ml.

Marking of milk fat globules with lectin-labelled gold granules MFG (0.05 ml, 5~ 10’ globules) were incubated for 2 h at 25°C with an excess of lectin-labelled Au III (ConA, 0.4 ml; WGA, 0.9 ml; SBA, 0.7 ml). The volume was adjusted to 1 ml with buffer A (SBA and WGA) or buffer B (ConA). MFG (0.5 ml; 5x 108)globules were also incubated with WGA-Au11 (0.6 ml). Control experiments were performed in the presence of 5 mg methyl

Location

of glycoproteins

cr-n-mannopyranoside (ConA), 5 mg N-acetyl-ogalactosamine (SBA) and 2 mg N-acetyl chitopentaose (WGA). Due to the density of gold, MFG marked with WGA and SBA-Au III were recovered in the pellet by centrifugation (15 set, 700 g) washed twice with buffer A and suspended in water (0.1 ml). When incubated with ConA-Au111 or in control experiments, MFG floated on the buffer (2 000 g, 5 min) which was sucked off and the globules were washed as above.

Marking of milk fat globule membranes with lectin-labelled gold granules MFGM (0.1 ml) were incubated for 2 h at 25°C with WGA-Au I (0.15 ml), WGA-, ConA- and SBA-Au II (0.15 ml), or a 1: 1 mixture of WGA-Au1 and SBAAu II (0.3 ml) in a total volume of 0.5 ml (buffer A). Control experiments were performed in the presence of 2 mg N-acetyl chitopentaose (WGA), 10 mg Nacetyl-o-galactosamine (SBA) and 10 mg methyl a-Dmannopyranoside (ConA). Marked MFGM were centrifuged at 700 g for 1 min and washed twice with buffer A.

Preparation

for SEM

Marked MFG were fixed for 1 h at 25°C in 0.05 M cacodylate buffer, pH 7.0 containing 2% 0~0,. No difference-was observed by SEM examination when MFG were fixed for 48 h in the same conditions. Fixed MFG were washed with water (4X) and 1 drop of susnension was deposited on SEM aluminium stubs. The specimens were air-dried and examined without metal coating in a Cambridge S 4-10 Stereoscan, operated at an accelerating voltage of 30 kV.

Preparation

for TEM

Marked MFG were embedded in tiny agar microcansules according to the method of Salyaev [20]. The capsules were fixed with glutaraldehyde and 0~0, and stained with uranyl acetate as described by Bauer [21]. Marked MFGM were embedded in gelatin microcapsules following the same procedure and were fixed only in 0~0, [21]. The microcapsules were finally embedded according to Spurr [22].

RESULTS Scanning electron microscopy of lectin-marked milk fat globules

Almost all lipids in milk occur in the form of globules having a size range of 0.1-20 pm in diameter[23]. When bovine MFG were incubated with WGA-Au III, the marker was found distributed in clusters over the whole surface of small and large MFG (figs 1, 2). Control ex-

on milk fat globule membrane

363

periments in the presence of N-acetylchitopentaose, a potent inhibitor of WGA [24] indicated that non-specific adsorption was very low (fig. 3). Human MFG from a blood donor 0 were marked to a small extent with ConA-Au III (fig. 4). No marking was detected with ConA on neuraminidase-treated MFG. When the same MFG were marked with WGA-AuIII, marking was dense, whether MFG had been treated with neuraminidase or not (figs 5, 6). Occasionally, some areas of the globule were not marked indicating a loss of the membrane (figs 5, 6, arrow). However, when MFG were incubated with SBA-AuIII, marking was increased by neuraminidase treatment (figs 7, 8). The same results were obtained with MFG from a blood donor A. Non-specific adsorption of SBA-Au111 was very low in control experiments . Marking of bovine MFG with WGAAu III was practically abolished in the presence of SBA (1 mglml). Quick agglutination of MFG with SBA was noticed. No marking of bovine MFG was obtained with peanut lectin, anti-H lectin and Bandeiraea simplicifolia lectin (Au III). Thin sections of milk fat globules marked with WGA-Au ZZ

Bovine MFG marked with WGA-Au11 were examined after thin sectioning (figs 9, 10). Marking was very dense on the unit membrane (fig. 9, arrow) and on areas where the unit membrane appeared to have been lost (fig. 9, double arrow). Vesicles still attached to MFG were also marked (fig. 10, arrows). Milk fat globule membranes marked with lectin-labelled gold granules

Isolated bovine MFGM were weakly marked with ConA-Au II (fig. 11) and more Exp Cell Res 109 (1977)

364

Horisberger, Rosset and Vonlanthen

Figs 1-3. Scanning electron microscopy of milk fat

globules marked with WGA. Figs I, 2. Bovine milk fat globules marked with WGAAu III. The marker is distributed in clusters over the whole surface. x7 Ooo. Fig. 3. A control experiment Bovine milk fat globules incubated with WGA-AU III in presence of N-acetyl-

chitopentaose. Non-specific adsorption is practically absent. X 11200. Figs 4-8. Scanning electron microscopy of milk fat globules marked with ConA, WGA and SBA. Fig. 4. Human milk fat globules marked with ConAAu III. Marking is weak and large area of the globule are not marked. X 11000.

densely (approximately to the same extent) with WGA- and SBA-Au II. A four-fold increase in the number of granules per unit area was noticed when MFGM were marked with WGA-Au1 instead of WGAAu II. MFGM were marked simultaneously

with WGAI and SBAII (fig. 12). When compared with MFGM marked either with WGA-Au1 or SBA-AuII, the density of marking was similar. Control experiments indicated that non-specific adsorption was absent.

Exp Cell Rrs 109 (1977)

Location of glycoproteins on milk fat globule membrane

365

Fig.

5. Human milk fat globules marked with WGAAuIII. Some areas are not marked (figs 5, 6, arrow) indicating a loss of the membrane. x 5 800. Fig. 6. Same as fig. 5, except that the globules were treated with neuraminidase. x 11700.

Fig. 7. Human milk fat globule marked with SBAAu III. x 15000. Fig. 8. Same as fig. 7, except that the globules were treated with neuraminidase. The density of marking was increased when compared with fig. 7. X 15000.

Contrary to MFG incubated with WGAAu111 in the presence of SBA, marking of MFGM with WGA-Au1 in the presence of SBA (1 mglml) was not prevented, although MFGM were readily agglutinated with SBA. MFGM marked with WGA I and SBA II

were also examined after thin sectioning (fig. 13). Both markers appeared to be located on one side of the membrane.

24-771804

DISCUSSION In the fluid mosaic model of membrane [25] some proteins and glycoproteins extend Exp

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Horisherger,

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completely through the bilayer while others are partially embedded in or on the membrane. Apparently all sugar residues of glycoproteins are located on the external surface of the membrane. Despite the fluidity of the lipids of milk fat droplets, MFG marked with lectinlabelled gold granules could be observed by SEM when they were properly fixed (figs l-8). Since these markers cannot penetrate the membrane, this demonstrates that part or all of the carbohydrate portion of MFGM glycoproteins are located on the external surface of the membrane. As similar results were obtained with erythrocytes [17], this indicated once more that the unit membrane of MFG is a true biological membrane. The occasional presence of large unmarked areas of MFG (figs 5, 6, arrow) could confirm that during milk ejection, membrane fragments desorb from the globule with a subsequent partial resorption of milk serum proteins [26]. These unmarked areas cannot be attributed solely to a loss of the unit membrane since small areas of MFGM, where the unit membrane was absent, were also marked (fig. 9). In membrane glycoproteins, sialic acid occupies a non-reducing terminal position and is linked either to D-g&rCtOSe or Nacetyl-D-galactosamine [27] and these sugars are also part of the cell surface receptors for SBA [28]. Sialoglycoproteins isolated from human and bovine milk have been shown to contain alkali labile tetrasaccharides whose structure has been proposed to be N-acetyl-neuraminyl(23)/3-Dgalactosyl( 1-3)-[N-acetylneuraminy1(2-6)]N-acetyl-D-galactosamine [29]. As most of the sialic acid can be released from MFG by neuraminidase without disrupting the fat globule [30], it was expected that neuraminidase-treated MFG would bind more SBA-Au III (fig. 8) than intact MFG (fig. 7). Exp Cell Res 109 (1977)

This increase was not due to the appearance of new SBA receptors on gangliosides containing sialic acid since MFG membrane proteins mask them from neuraminidase attack [3 I]. Despite the fact that MFG contain ConA receptors disposed externally on their surface [9] MFG were not well marked with ConA-Au III (fig. 4). This was analogous to human erythrocytes which have 1.2~ lo5 ConA binding sites per cell [32] but are not marked with ConA-Au III (unpublished observation). It is therefore likely that glycoproteins having receptors for ConA are not accessible to a large size marker such as ConA-Au111 (50 nm 0). This masking effect must be attributed to other proteins or glycoproteins (see below) and not to the negative charge of the MFG surface due to sialic acid since treatment of MFG by neuraminidase did not improve the marking. Similar observations were found with peanut lectin and anti-H lectin. The unsubstituted disaccharide /3-D-galactosyl(1,3)-N-acetyl-D-galactosamine was found to be present to a different extent in MFGM glycoproteins [29] and would act as a receptor for peanut lectin. However, no marking was obtained with this lectin. Fips 9-13. Electron microscopy of milk fat globule

membranes marked with WGA; ConA and SBA. Fig. 9. Thin section of bovine milk fat globule marked

with WGA-AuII. Dense marking is observed on the unit membrane (arrow) and in areas where the unit membrane is absent (da&e arrow). x90000. Fig. 10. Same as fig. 9. The degeneration of the unit membrane bv vesiculation is seen. The vesicles are marked (m&v). x90000. Fia. II. Isolated bovine milk fat globule membrane marked with ConA-Au II. Marking $ weak and distributed in patches. x48000. Fip. 12. Isolated bovine milk fat alobule membranes m&ked simultaneously with *GA-Au I (smaller granules) and SBA-Au 11 (larger granules). The Iectin receptors appear to be clustered. ~48000. Fig. 13. Same as fig. 12. The lectin receptors are located almost exclusively on one side of the membrane (arrow). ~85000.

Location k

of glycoproteins

on milk fat globule,membrane

367

r

_

0.1

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Horisherger,

Rosset and Vonlanthen

Sialoglycoproteins containing L-fucose (a receptor for anti-H lectin) have also been found in MFGM [29, 301. Again, L-fucose was not marked with this lectin. In view of these results, the absence of marking of MFG with Bandeiraea simplicifolia lectin does not rule out the presence of a-galactosyl unit in MFGM glycoproteins. As many glycoproteins from MFGM contain receptors for WGA and SBA [6, 29, 301 it is impossible at present to know which fractions are receptors for both lectins. WGA has been reported to bind non-specifically to N-acetyl-neuraminic acid [33]. This possibility was excluded in the case of MFG since total inhibition of WGA marking was obtained in the presence of N-acetyl-chitopentaose (fig. 3) and WGA marking was not decreased when MFG were treated with neuraminidase. When MFG were incubated with soluble SBA, WGA-Au111 marking was almost completely absent. This would indicate either that receptors for both lectins are on the same glycoprotein or rather (see below) that glycoproteins binding SBA or WGA are close to each other. Bound SBA would therefore hinder the access of the large size marker WGA-Au III to its receptor. Examination of thin sections of bovine MFG marked with WGA-Au II showed that the membrane was marked continuously when the unit membrane was not present (fig. 9, double arrow). It is known that after secretion some of the unit membrane is lost by vesiculation into the milk serum [3, 41, a process which is clearly seen in fig. 10. As observed by Wooding [3], these vesicles contain a dense material which is thought to derive from the cytoplasm of the lactating cell [3-51. Isolated MFGM were marked more densely with ConA-Au11 (fig. 11) than MFG with ConA-Au III (fig. 4) which would indiE.xp Cd

Res 109 (1977)

cate that access of the small size marker Au11 (26 nm 0) to its receptor is less hindered than that of Au III (50 nm 0) by sialoglycoproteins which protrude from mammalian cell surface [34]. Double marking of isolated bovine MFGM with WGA-AU I and SBA-Au II is shown in fig. 12. Although MFGM were prefixed with glutaraldehyde the receptors for both lectins appeared clustered at this scale of magnification. Since the density of both markers was similar to that obtained in separate experiments, it is thought that receptors for both lectins are located on different glycoproteins. The low density of marking of fig. 12 when compared to fig. 9 was probably due to the known loss of glycoproteins during the isolation of MFGM [l, 351. Therefore caution should be exercised in extrapolating results obtained from isolated MFGM to MFG. Despite the rather low density of marking of MFGM marked with WGA-Au1 and SBA-Au11 (fig. 12), lectin receptors appeared to be located in thin sections only on one side of the MFGM as found with other biological membranes (see review in ref. [36]). In conclusion, the method employed in this report provides a specific way to study the topological distribution of lectin receptor sites on MFG and on MFGM. The present results indicate that MFGM is a true biological membrane containing glycoproteins, part of which is not accessible to lectin-labelled gold granules. Very similar results obtained with human erythrocytes will be discussed elsewhere. No striking difference was observed using either bovine MFG or human MFG from two different blood groups. We are greatly indebted to the hospital La Providence (La Tour-de-Peilz, Switzerland) for the human milk

Location of glycoproteins on milk fat globule membrane samples and to Mrs M. Weber for the photographic work.

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19. Keenan, T W, Morre, D, Olson, D E, Yunghans, W N & Patton, S, J cell biol44 (1970) 80. 20. Salyaev, R K, Fourth cur reg donf electron microscopy, Rome 2 (1968) 37. 21. Bauer, H, J dairy sci 55 (1972) 1375. 22. Spurr, A R, J ultrastruct res 26 (1969) 3 I. 23. Walstra, P, Netherlands milk dairy j 23 (1969) 99. 24. Goldstein, 1 J, Hammerstrom, S & Sundblad, G, Biochim biophys acta 405 (1975) 53. 25. Tip,, S J & Nicolson, G L, Science 175 (1972) 26. Henson, A F. Holdsworth. G & Chandan. R C. J dairy sci 54 (1971) 1752. 27. Spiro, R G, Ann rev biochem 39 (1970) 599. 28. Lis, H, Sela, B, Sachs, L & Sharon, N, Biochim biophys acta 211 (1970) 582. 29. Glockner, W M, Newman, R A, Dahr, W & Uhlenbruck, G, Biochim biophys acta 443 (1976) 402. 30. Harrisson, R, Higginbotham, J D & Newman, R, Biochim biophys acta 389 (1975) 449. 31. Tomich, J M, Mather, I H & Keenan, T W. Biochim biophys acta 433 (1976) 357. 32. Schnebli. H P & B&hi. T. EXD cell res 91 (1975) 175. 33. Greenaway, P J & Le Vine, D, Nature new biol 241 (1973) 191. 34. Pinto da Silva, P & Nicolson. G L. Biochim biophys acta 363 (1974) 3 11. 35. Anderson, M & Brooker. B E. J dairv sci 58 (1975) 1442. 36. Nicolson, G L, Biochim biophys acta 457 (1976) 57. Received April 27, 1977 Accepted May 27, 1977

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