Comparison of lectin receptor and membrane coat-associated glycoproteins of milk lipid globule membranes

Comp. Biochem. Physiol.. Vol. 63B. pp. 137 to 145

0305-0491/79/0105-0137502.00/0

© Pergamon Press Ltd 1979. Printed in Great Britain

COMPARISON OF LECTIN RECEPTOR A N D MEMBRANE COAT-ASSOCIATED GLYCOPROTEINS OF MILK LIPID GLOBULE MEMBRANES LYNNE R. MURRAY, KAREN M. POWELL, M. SASAKI,W. N. EIGEL and T. W. KEENAN Mammary Biology Laboratory, Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907 U.S.A. (Received 19 September 1978) Abstract--1. Glycoproteins of bovine (Bos taurus) and human (Homo sapiens) milk lipid globule membranes were characterized by ability to bind lectins after electrophoretic separation. 2. Seven lectin receptor glycoproteins were detected in bovine and five in human milk lipid globule membranes. Bovine and human globule membrane glycoproteins differed in ability to interact with certain lectins. 3. Two major nonionic detergent insoluble glycoproteins were present in bovine and human lipid globule membrane; these constituents had apparent molecular weights of 155,000 and 69,000. Detergentinsoluble polypeptides with similar or identical electrophoretic mobilities were found in milk lipid globule membranes from four other species, rat (Rattus norvegicus), sheep (Ov/s aries), pig (Sus scrofa) and goat (Capra hircus). Tryptic peptide mapping revealed these polypeptides to be nonidentical among species.

INTRODUCTION

Milk fat globule membrane (MFGM), a trilaminar, unit-like membrane which surrounds lipid globules in milks of all species examined to date, is derived primarily from the apical plasma membrane of mammary alveolar epithelial cells (reviews, Patton & Keenan, 1975; Anderson & Cawston, 1975; Wooding, 1977). While MFGM has become increasingly popular as a model for studies of plasma membrane properties, most of the research has been conducted with MFGM of bovine origin. Several groups have characterized polypeptides of bovine MFGM with respect to electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels (Kobylka & Carraway, 1972; Anderson et al., 1974; Kitchen, 1974; Mather & Keenan, 1975), a technique which allows estimation of molecular weights. While these groups are in agreement as to the number and molecular weights of major MFGM polypeptides, the number of periodateSchiff positive glycoproteins of this membrane has been variously reported as from 4 to 8. In contrast to bovine MFGM, only two studies of human MFGM polypeptides have been reported (Martel et al., 1973; Freudenstein et al., 1978). To our knowledge the only study of glycoproteins of human MFGM has been that of Martel et al. (1973), who found only two glycoprotein staining bands in electrophoretograms. Freudenstein et al. (1978) reported that polypeptides of the detergent-insoluble coat fraction associated with human MFGM contained several carbohydrates. Previous investigators have shown that lectins interact with constituents of MFGM. Horisberger et al. (1977) used lectin-gold granule complexes to study the location of glycoproteins in bovine and human MFGM. Keenan et al. (1974) assayed the ability of

isolated MFGM and endomembranes from bovine mammary gland to bind Concanavalin A. Newman & Uhlenbruck (1977) used various lectins to establish the presence of the Thompsen-Friedenreich antigen in bovine MFGM; this antigen was not detected in human MFGM. In this report we present results of comparative studies on glycoproteins of bovine and human MFGM. These glycoproteins have been characterized by their ability to bind various lectins after electrophoretic separation. In addition, polypeptides of molecular weights 155,000 and 69,000, which are major insoluble proteins associated with internal coat structures of MFGM (Keenan et al., 1977; Freudenstein et al., 1978) have been compared for identity by peptide mapping. This comparison was made with MFGM coat fractions from cow, goat, sheep, sow, rat and human milks. MATERIALS AND METHODS

Fresh bovine milk was obtained from animals of the Holstein breed. Human milk was from volunteers in established lactation. Milk samples were also obtained from a Merino ewe, a Yorkshire sow, a Nubian goat and several albino Wistar rats; all were in established lactation at the time of sample collection. Concanavalin A and trypsin (type X, DCC-treated) were from Sigma, St. Louis, Mo, U.S.A. Wheat germ agglutinin and soybean agglutinin were from Miles-Yeda, Rehovot, Israel. Ricinus communis-120 lectin was from P-L Laboratories, Milwaukee, Wis., U.S.A. Carrier free Na12Sl (15 mCi//~g I) was from Amersham, Arlington Heights, Illinois, U.S.A. MFGM was prepared from washed cream as described (Mather & Keenan, 1975). Total milk lipid globule proteins were obtained by preparation of an acetone-diethyl ether powder of washed cream. Fractions enriched in MFGM internal coat-associated polypeptides were prepared by

137

138

kYNNE R. MURRAY et al.

extracting M F G M with 11;~i Triton X-100 in 10 mM Tris-HC1, pH 8 (1 ml/mg proteint for 1 hr at 3 7 C (Freudenstein et al., 1978). Insoluble coat material was collected by centrifugation at 100,000g for 60 min and washed with 10 m M Tris-HCl, pH 8. For polyacrylamide gel electrophoresis, both M F G M and coat associated polypeptides were dissolved in sodium dodecyl sulfate under reducing conditions (Mather & Keenan, 1975) and were separated in 8"~ polyacrylamide slab gels (Weber & Osborn, 1969). Gels were stained with Coomassie blue and destained by diffusion. Glycosylated proteins were detected with the periodate-Schiff reagent (Fairbanks et at., 1971). For lectin staining, gels were fixed by the method of Olden & Yamada (1977), rinsed with several changes of calcium- and magnesium-free phosphate buffered saline (PBS), incubated with the appropriate ~251-1ectin in PBS (10 #g//ml) overnight and again rinsed with several changes of PBS to remove unbound lectin. Gels were then stained

with Coomassie blue, destained, dried with a slab gel dryer (Bio-Rad Laboratories. Richmond, Cal., U.S.A3 and exposed for 2 6 days to Kodak Royal Blue X-Omat X-ray film. Two methods were used to check for nonspecific lectin binding. Gels were incubated with lectin in the presence of 0.1 M hapten sugar for the particular lectin (~.-methyl mannoside for Concanavalin A: N-acetylglucosamine for wheat germ agglutinin: fl-D-galactose for Ricinus communis lectin: and N-acetyl-galactosamine for soybean agglutinin). Alternatively, gels were labelled with lectin and then were incubated with 0.1 M hapten sugar in PBS overnight. Lectins were iodinated by the chloramine T method (Chang & Cuatrecasas, 19761. Concanavalin A was separated from the reaction mixture by affinity chromatography over Sephadex G-25 (Chang & Cuatrecasas, 1976t. Free iodine and salts were removed from the other lectins by exhaustive dialysis against PBS at 4 ('. Lectins were stored at 4 C in PBS containing 0.1'!~, sodium azide.

Fig. 1. Electrophoretic pattern of bovine and human milk lipid globule polypeptides. The gel was stained with 0.05°,~, Coomassie blue. Lane A, bovine milk lipid globule acetone powder; B, bovine MFGM; C, Triton-insoluble residue of bovine M F G M ; D, human milk lipid globule acetone powder; E, Triton-insoluble residue of human M F G M : F, standard proteins myosin (m), bovine serum albumin (b), ovalbumin (o), and cytochrome c (cl. Positions of bands 3, 12, 16 and 18 are indicated. Arrows denote positions of periodate-Schiff staining glycoproteins I to VII from top to bottom, respectively.

Glycoproteins of milk lipid globule membranes For peptide mapping, bands of interest were sliced from Coomassie blue stained gels, protein was eluted and recovered (Bray & Brownlee, 1973). Samples were iodinated, oxidized with performic acid and digested with trypsin (1 mg/ml) for 16 hr at 37°C; radioactive contaminants were then removed on small columns of Sephadex G-25 (Bray & Brownlee, 1973) and peptides were separated in two dimensions by thin-layer chromatography and electrophoresis. For this, peptides were dissolved in 1% ammonium hydroxide and spotted near one edge of a plastic sheet coated with silica gel G (Eastman Chromagram sheet without fluorescent indicator). Chromatograms were developed in n-propanol-28% ammonium hydroxide (7:3, v/v). When solvent reached the top, sheets were air-dried and subjected to electrophoresis in the perpendicular dimension for 2.5 hr at 500 volts in pyridine-acetic acidwater (1:10:289, v/v). After drying, separated p~?tides were detected by autoradiography. One-dimensional peptide maps were obtained by the chromatographic step alone.

RESULTS AND DISCUSSION Sodium dodecyl suifate-polyacrylamide gel electrophoretic patterns obtained with bovine and human M F G M polypeptides are compared in Fig. 1. As noted previously for bovine M F G M (Mather & Keenan, 1975; Mather et al., 1977; cf. also Fig. 1), there is a selective loss of certain lipid globule polypeptides and corresponding enrichment of others during lysis of both bovine and human milk lipid globules and subsequent centrifugal recovery of MFGM. Many polypeptides with common mobilities were shared by bovine and human M F G M (Fig. 1). This was most evident when the Triton X-100 insoluble residues of M F G M from both sources were compared (Fig. 1). For convenience, major bovine M F G M polypeptides are numbered according to Mather & Keenan (1975); those in human M F G M with similar mobilities will be referred to by the same numbers with the prefix H. Positions of periodateSchiff staining glycoproteins are indicated in Fig. 1 and are designated by Roman numerals as indicated. Periodate-Schiff staining bands I and II of bovine M F G M do not stain with Coomassie blue; bands III, IV, V, VI and VII correspond to Coomassie blue stained bands 3, 10, 12, 16 and 18, respectively (cf. also Mather & Keenan, 1975). Periodate-Schiff staining bands of human M F G M correspond to Coomassie blue bands H3, H10, H12, HI6 and H18 and are designated as H-I, H-II, H-Ill, H-IV and H-V, respectively. With both bovine and human MFGM, Triton extraction led to large enrichments of bands 3 (apparent molecular weight 155,000) and 12 (apparent molecular weight 68,000) in the insoluble residue. Concanavalin A, a lectin specific for ~t-D-mannose or ~t-D-glucose residues (Nicholson, 1974), was bound by all seven glycoproteins (representing bands I to VII) of bovine M F G M (Fig. 2). Nearly all of these bands were also stained by Concanavalin A in Tritoninsoluble residues of bovine MFGM, but bands I, II, IV and VII were more lightly stained and band V was more intensely stained than in untreated membranes. Triton extraction appeared to remove nearly all Concanavalin A-binding polypeptides from bands VI and VII. Human M F G M polypeptides in the regions of bands I, II and III bound Concanavalin

139

A to a moderate extent; a broad region (which appeared to be a doublet) around band IV was densely stained with this leetin (Fig. 2). Triton extraction of human M F G M removed Concanavalin A receptor glycoproteins from bands I and III (Fig. 2). In all cases, Concanavalin A binding appeared to be specific in that binding was not observed in the presence of or-methyl mannoside and bound lectin could be displaced on incubation of Concanavalin A treated gels with this carbohydrate. Wheat germ agglutinin, a lectin specific for N-acetylglucosamine residues (Nagato & Burger, 1974), was bound by all seven glycoproteins of bovine M F G M (Fig. 3). Triton extraction intensified the staining of bovine bands I and V and removed the band VI material which bound this lectin. Two diffuse bands of human M F G M were labelled with wheat germ agglutinin (Fig. 3). Band H-II was faintly stained whereas a broad band, representing H-IV and H-V, was more intensely stained. A similar or identical pattern was observed with the Triton-insoluble residue of human M F G M (not shown). Soybean agglutinin, which is specific for N-acetylgalactosamine and D-galactose residues (Lis & Sharon, 1972), was bound by all seven electrophoretically separable glycoproteins of bovine M F G M (Fig. 4). After extraction with Triton, only bands I, III, IV, V and VII were stained in the insoluble residue; soybean agglutinin-binding glycoproteins of bands II and VI were apparently completely solubilized by this

Fig. 2. Concanavalin A binding to glycoproteins of MFGM. Lane A, standards showing staining of ovalbumin; B, bovine MFGM; C, Triton-insoluble residue of bovine MFGM; D, human MFGM; E, Triton-insoluble residue of human MFGM.

140

LYNNE R. MURRAYet al. In summary, several lectins, with different carbohydrate specificities, were found to interact with at least seven and five electrophoretically distinct glycoproteins in bovine and human MFGM, respectively. Lectin-binding to these glycoproteins was specific in that, in all cases, binding was not obtained in the presence of hapten sugar. In addition to differing in total numbers of lectin receptor glycoproteins, bovine and human MFGM glycoproteins were observed ro differ in ability to interact with lectins. The lectins Concanavalin A, wheat germ agglutinin and soybean agglutinin interacted with all seven bovine MFGM glycoproteins. In contrast, with human MFGM, Concanavalin A interacted with four of the five periodateSchiff staining glycoproteins, wheat germ agglutinin was bound by only two or three glycoproteins and soybean agglutinin did not interact with any glycoproteins to a detectable extent. These results imply that there are differences in carbohydrate accessibility or composition in bovine and human MFGM glycoproteins. Differential removal of both bovine and human MFGM glycoproteins by Triton extraction was also observed using lectin binding and autoradiography. For example, Triton extraction of bovine MFGM

Fig. 3. Wheat germ agglutinin binding to glycoproteins of MFGM. Lane A, bovine MFGM; B, human MFGM; C, Triton-insoluble residue of bovine MFGM. The pattern obtained with the Triton-insoluble residue of human MFGM was identical to that shown in lane B.

treatment. Triton extraction greatly diminished the relative staining intensity of band IV. Band II1 was but weakly stained with soybean agglutinin in both total and extracted bovine MFGM. Human MFGM glycoproteins were not stained with soybean agglutinin either before or after Triton extraction. In contrast, Horisburger et al. (1977), who used electron microscopy to study the interaction of lectin-gold complexes with milk lipid globules, observed binding of soybean agglutinin by human milk lipid globules. Our observation with soybean agglutinin was surprising in that appreciable amounts of protein-bound galactose and N-acetylgalactosamine are present in Triton-insoluble residues of human MFGM (Freudenstein et al., 1978). Apparently these carbohydrate residues are structurally inaccessible to this lectin. In preliminary trials. R i c i n u s c o m m u n i s lectin (flD-galactose specific) was found to interact with all seven glycoproteins of bovine MFGM and the five periodate-Schiff positive glycoproteins of human MFGM (not shown).

Fig. 4. Soybean agglutinin binding to glycoproteins of MFGM. Lanes A and D, Triton-insoluble residue of bovine MFGM; B, human MFGM: C, bovine MFGM.

Glycoproteins of milk lipid globule membranes

141

Fig. 5. Electrophoretic comparison of polypeptides in Triton-insoluble residues of MFGM from human (Lane B), rat (C), pig (D), sheep (E), goat (F) and bovine (G) milks. Lanes A and H, standard proteins myosin (o), bovine serum albumin (b), ovalbumin (o) and cytochrome c (c). The positions of bands 3 and 12 are indicated by arrows. The gel was stained with 0.05~o Coomassie blue.

removed glycoprotein receptors for Concanavalin A, wheat germ agglutinin and soybean agglutinin migrating with band VI. This extraction removed soybean agglutinin receptor, but not receptors for other lectins, from band II. This suggests the occurrence of more than one glycoprotein in band II. Of particular interest was the staining of bovine bands III and V and human bands H-I and H-Ill with lectins both before and after Triton X-100 extraction. These glycoproteins, which corresponded in electrophoretic mobility to Coomassie blue staining bands 3 (bovine III and human H-I) and 12 (bovine V and H-Ill) of bovine MFGM, have been shown to be the major polypeptide constituents of the coat material associated with bovine and human MFGM (Freudenstein et al., 1978). This coat material is associated with the inner face (the membrane face which borders the lipid globule core) of MFGM. Band 12 has been purified from bovine MFGM and rabbit antisera to this material cross reacts with detergent extracts of sheep, goat and pig MFGM, but not with extracts of rat or human MFGM (Freudenstein et al., 1978). This suggested the presence of a common polypeptide or group of polypeptides in MFGM from several species and we have studied this further. When Triton insoluble residues of rat, human, pig, sheep,

and goat MFGM were separated on polyacrylamide gels, bands with mobilities similar to bovine bands 3 and 12 were observed in all preparations (Fig. 5). These bands were recovered, radioiodinated, and tryptic peptides were prepared and separated. Onedimensional peptide maps revealed some homology of bands 3 and 12 among the species (not shown). With band 3, pig, goat, sheep and rat appeared closely related and different from cow and human. With band 12, human and rat were similar to each other and different from the similar peptide patterns observed with pig, sheep, goat and cow. When tryptic peptides of band 3 were separated in two dimensions, it was obvious that polypeptides of this band were not identical among any of the species studied (Fig. 6). Differences in peptide maps were greater in all cases than would be expected from only limited amino acid substitutions or differing degrees of glycosylation. Our interpretation of the degree of relatedness of band 3 among the species studied is summarized in Table 1. Band 12 polypeptides were also nonidentical among the six species studied (Fig. 7). Again, differences were larger than would be expected from limited amino acid substitutions or different degrees of glycosylation. Human and rat band 12 were most

LYNNE R. MURRAY et al.

142

F

tlk

Fig. 6. Two-dimensional tryptic peptide maps of (125I)-labelled band 3 from Triton-insoluble residues of human (A), rat (B), pig (C), sheep (D), goat (E) and bovine (F) M F G M . Origins are at the lower left. Chromatography was in the vertical direction and electrophoresis was from left (+) to right ( - ) .

Glycoproteins of milk lipid globule membranes

143

!r

Fig. 7. Two-dimensional tryptic peptide maps of (l~sI)-labelled band 12 from Triton-insoluble residues of human (A), rat (B), pig (C), sheep (D), goat (E) and bovine (F) M F G M . Origins are at the lower left. Chromatography was in the vertical direction and electrophoresis was from left ( + ) to right ( - ) .

144

LYNNE R. MURRAY et al. Table I. Similarity of peptide maps for milk fat globule membrane band 3 material from several species*

Pig

Goat

Pig

+4-+

Goat

++

+-~

Sheep

++

++

Sheep

4-+

Human

Human

Cow

Rat

++

-

+

+

q-+

-

+

+

-

+

+

+

+

~

-

-

+++

Cow

+

+

+

+

Rat

+

+

+

+

+++

+

+

+'4"+

* Summarized from Fig. 6. The degree of homology of peptide maps among species is expressed qualitatively as--(little or no homology) through + + + (closely similar or identical).

Table 2. Similarity of peptide maps for milk fat globule membrane band 12 material from several species* Human

Human

+++

Rat

Pig

+

+++

Sheep

Goat

-

-

-

-

-

-

++

+

+

-+-+4-

+

+

Rat

+

Pig

-

+++

Sheep

-

++

Goat

-

+

+

+4-+

+

+

4~

Cow

Cow

4-+ 4-++

* Summarized from Fig. 7. The degree of homology of peptide maps among species is expressed qualitatively as~(little or no homology) through + + + (closely similar or identical).

similar to each other and distinctly different from the other species. Similarly, pig and sheep b a n d 12 materials yielded somewhat related peptide maps, as did goat a n d bovine b a n d 12 materials. Interpretation of the degree of relatedness of b a n d 12 a m o n g species is given in Table 2. Thus, while various degrees of homology were shared in bands 3 and 12 polypeptides between species, it is apparent that they are nonidentical a m o n g the species studied. This does not rule out the possibility that these polypeptides serve the same, albeit unknown, functions in these species. Acknowledgements--This research was supported by grants GM 23889 from the National Institute of General Medical Science and PCM75-11908 from the National Science Foundation. Purdue University Agricultural Experiment Station Journal Paper No. 7327. REFERENCES

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