Sialoglycopeptides from bovine milk fat globule membrane.

Milk fat globule membrane was shown to contain sialic acid, all of which could be released without disruption of the fat globule. Sialoglycopeptides were cleaved from the surface of intact fat globules by Pronase and fractionated on Sephadex G-50. Further fractionation of the major sialoglycopeptide peak on DEAE-Sephadex gave two groups of sialoglycopeptides eluted with 0.1 M NaCl (Group A) and 0.5 M NaCl (Group B), respectively. Refractionation gave a major sialoglycopeptide from each of the two groups together with a total of three minor sialoglycopeptides. All five sialoglycopeptides eluted as single peaks using shallow salt gradients on DEAE-Sephadex and contained a hydrophilic peptide chain together with galactose, mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid. Glycopeptides of Group A but not Group B contained fucose. The major sialoglycopeptide of Group B released 35% of its hexose and hexosamine on treatment with alkaline borohydride leaving a sialoglycopeptide which had reduced serine and threonine and elevated alanine levels and in addition contained 2-aminobutyric acid. An oligosaccharide fraction containing N-acetylgalactosaminitol, galactose and sialic acid in a molar ratio of 1:1:2 was partially characterised from the clevage mixture. The major sialoglycopeptide of Group A had a more complex carbohydrate structure and showed no released carbohydrate on treatment with alkaline borohydride. The sialoglycopeptides of milk fat globule membrane show many similarities with those of erythrocyte membrane and have a potential use in comparative and structural studies.     

Sialoglycopeptides from bovine milk fat globule membrane

Milk fat globule membrane was shown to contain sialic acid, all of which could be released without disruption of the fat globule. Sialoglycopeptides were cleaved from the surface of intact fat globules by Pronase and fractionated on Sephadex G-50. Further fractionation of the major sialoglycopeptide peak on DEAE-Sephadex gave two groups of sialoglycopeptides eluted with 0.1 M NaCI (Group A) and 0.5 M NaCI (Group B), respectively. Refractionation gave a major sialoglycopeptide from each of the two groups together with a total of three minor sialoglycopeptides. All five sialoglycopeptides eluted as single peaks using shallow salt gradients on DEAE-Sephadex and contained a hydrophilic peptide chain together with galactose, mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid. Glycopeptides of Group A but not Group B contained fucose.The major sialoglycopeptide of Group B released 35 % of its hexose and hexosamine on treatment with alkaline borohydride leaving a sialoglycopeptide which had reduced serine and threonine and elevated alanine levels and in addition contained 2-aminobutyric acid. An oligosaccharide fraction containing N-acetylgalactosaminitol galactose and sialic acid in a molar ratio of 1 : 1 : 2 was partially characterised from the cleavage mixture.The major sialoglycopeptide of Group A had a more complex carbohydrate structure and showed no released carbohydrate on treatment with alkaline borohydride.The sialoglycopeptides of milk fat globule membrane show many similarities with those of erythrocyte membrane and have a potential use in comparative and structural studies.     

Alkali-labile oligosaccharides from bovine milk fat globule membrane clycoprotein

Phenol extraction of bovine milk fat globule membrane gave a glycoprotein fraction which, in sodium dodecyl sulphate electrophoresis, showed three major bands, all staining for both protein and carbohydrate. Alkaline borohydride treatment and desialylation of the glycoprotein fraction released the reduced disaccharide β-d-galactosyl(1 → 3)-$\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ (T-antigen), which was identified by gas chromatography using a standard. All of the disaccharide units in the native glycoprotein were shown to be substituted by sialic acid, and a tetrasaccharide containing the disaccharide plus two molecules of sialic acid was isolated following alkaline borohydride treatment of the glycoprotein and gel filtration. Periodate oxidation of native and desialylated glycoprotein, together with paper chromatography of alkali degraded oligosaccharide fragments, indicated that the major alkali-labile oligosaccharide of the glycoprotein fraction is a tetrasaccharide containing $\text{β-}\text{d}\text{-galactosyl(1 → 3)}\text{-N-}\text{acetyl-}\text{d}\text{-galactosamine}$ substituted by sialic acid at position C3 of the galactosyl and position C6 of the $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ residue. Evidence was also obtained for the presence of small amounts of unsubstituted alkali-labile $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ linked directly to protein in the native glycoprotein.Serological evidence using agglutinins from Vicia graminea, Arachis hypogoea and human anti-T serum confirmed the presence in the native glycoprotein of a sialic acid substituted T-antigen. Similar evidence using agglutinins from Helix pomatia and Cepaea hortensis also confirmed the presence of terminal alkali-labile $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ in the native glycoprotein.     

Crossed immunoelectrophoresis of bovine milk fat globule membrane protein solubilized with non-ionic detergent.

Detergent solubilized bovine milk fat globule membrane material studied by crossed immunoelectrophoresis combined with histochemical techniques revealed four major protein complexes. All four were found to bind to concanavalin A and three were identified as sialoglycoproteins. Xanthine oxidase activity was associated with the non-sialoglycoprotein precipitate. Immunoabsorption with intact milk fat globules showed an internal location of the xanthine oxidase, whereas the three other main proteins plus Mg2+-ATPase and 5'-nucleotidase were disposed on the outer membrane surface. The major proteins from milk fat globule membrane and membrane material isolated from skim milk showed immunochemical identity.     

Characterisation of the surface of bovine milk fat globule membrane using microelectrophoresis

The nature of the ionogenic groups on the surface of the milk fat globule membrane was studied by microelectrophoresis of intact fat globules after chemical and enzymic modification. The changes in pH-mobility curves effected by formaldehyde and 2,4-dinitrofluorobenzene showed that the membrane surface contained amine groups. These were identified as arising from lysine and arginine by chromatography of their dinitrophenyl derivatives. The contribution of N-acetylneuraminic acid and phosphate to the surface charge was demonstrated by their specific removal by neuraminidase and phospholipase C, respectively. After removal of N-acetylneuraminic acid and phosphate, anionogenic effects remained which were attributed to protein carboxyl groups. These groups could be partially esterified using diazomethane. The effect of sodium dodecyl sulphate and of ionic strength on electrophoretic mobility indicated that the surface contains little neutral lipid and is predominantly ionogenic. The results obtained concerning the nature of the surface of the milk fat globule membrane support the hypothesis that the milk fat globule membrane originates from the plasmalemma of the mammary alveolar cell.     

Qualitative and quantitative profiling of the bovine milk fat globule membrane proteome

Milk is a biological fluid of unique quality and complexity. It has co-evolved with mammals and mankind to nourish offspring and contains macro- and micronutrients for growth and development of the newborn. The milk fat globule membrane (MFGM) represents an important milk fraction, which is rich in bioactive proteins. In order to better understand functionality of milk fractions and, thereby, enhance the benefits of milk products, detailed qualitative and quantitative protein knowledge of fractions such as MFGM is required.We report the qualitative and quantitative profiling of two MFGM-enriched milk fractions, a whey protein concentrate (WPC) and a buttermilk protein concentrate (BMP), as derived from three different analytical workflows. First, an LC-MS/MS-based shotgun approach revealed 244 protein identities in WPC and 133 in BMP, respectively, and provided an extensive characterisation of the protein content in those two fractions. Second, label-free profiling resulted in rapid and efficient semi-quantitative comparison and yielded valuable protein fingerprints. Third, absolute quantification of selected MFGM proteins was achieved by stable isotope dilution (SID)-MS, in combination with multiple reaction monitoring (MRM) detection. In summary, we provide new information on composition, quantity and possible health benefits of two MFGM-enriched milk fractions highly valuable for future nutritional applications.     

The sheep milk fat globule membrane proteome

Milk fat globule membranes (MFGM) are three-layered structures that enclose fat droplets, and are composed by an internal monolayer of endoplasmic reticulum origin, surrounded by a bilayer derived from the apical membrane of the lactating cell. In this work, an optimized protein extraction method was applied to sheep MFGM, and extracts were subjected to SDS-PAGE separation followed by shotgun LC tandem mass spectrometry (GeLC-MS/MS) for identification and characterization. In total, 140 unique sheep MFGM proteins (MFGMPs) were identified. All protein identification data were subjected to Gene Ontology (GO) classification for localization and function. Moreover, the relative abundance of all identified MFGMPs was estimated by means of the normalized spectral abundance factor (NSAF) approach, and GO abundance classes were obtained. The data gathered in this work provide a detailed picture of the proteome expressed in healthy sheep MFGs, and lay the foundations for future studies on sheep lactation physiology and on its alterations in pathological conditions.     

Selective extraction of marker enzymes of bovine milk fat globule membrane by nonionic detergents.

Large amounts (66-97%) of marker enzymes such as alkaline phosphatase, 5'-nucleotidase, phosphodiesterase I, and gamma-glutamyl transpeptidase of bovine milk fat globule membrane (MFGM) were selectively solubilized by nonionic detergents such as Triton X-100, Tween 20, Nonidet P-40, Liponox NCK, and Emulgen 109-P. On the other hand, the extractability of MFGM protein with these detergents was less than 50%. Judging from the recovery of total activity, it is likely that alkaline phosphatase, phosphodiesterase I, and gamma-glutamyl transpeptidase are activated by nonionic detergents, whereas 5'-nucleotidase is somewhat inhibited by the detergents, except for Tween 20, and acid phosphatase is strongly inhibited by all detergents. In addition, solubilization of the protein with the nonionic detergents was found to be somewhat selective by SDS-polyacrylamide gel electrophoresis. There was no appreciable difference between the five brands of nonionic detergents used as regards the extractability of protein and the enzymatic activity of the extracted marker enzymes of MFGM, except that the solubilizing ability of Tween 20 was relatively low.     

Studies on the structure of milk fat globule membrane.

Milk fat globule membrane was solubilized with sodium dodecyl sulfate and mercaptoethanol and the membrane proteins were separated by SDS-polyacrylamide gel electrophoresis. The membrane preparations contained three major size classes of polypeptide of 155,000, 62,500 and 43,500 daltons. At least five glycopeptides were separated of which two stained intensely with periodic acid-Schiff reagent, but poorly with coomassie blue. Trypsin hydrolysis of whole cream and isolated milk fat globule membrane revealed major differences in the rates of protein hydrolysis. Many of the membrane proteins of whole cream resisted proteolysis compared with the same proteins in the isolated membrane. Two glycopeptides were resistant to trypsin digestion in either preparation. Treatment of whole cream with neuraminidase led to the release of at least 70% of the protein-bound sialic acid. Whole cream and isolated membrane samples were iodinated with 125I in the presence of lactoperoxidase and hydrogen peroxide. The membrane proteins were significantly more accessible to lactoperoxidase-125I i in isolated membrane compared with the proteins of whole cream. Polypeptides of molecular weight 43,500 and approximately 48,000 daltons were predominantly labelled in whole cream and could be eluted from the fat globules with magnesium chloride (1.5m). The results strongly suggest that the proteins of milk fat globule membrane are asymmetrically arranged in the membrane and that most of the protein-bound sialic acid is present on the external surface of milk fat globules.     

Immunological relationship between the hydrophobic fraction of proteose-peptone and the milk fat globule membrane of bovine milk

The antigenic relationship between the milk fat globule membrane (MFGM) and the hydrophobic fraction of proteose-peptone (HFPP) was demonstrated, using a mono-specific anti-HFPP antibody.     

Glycosaminoglycans of the fat globule membrane of cow milk

Membranes of fat globules of cow milk contained 163 μg/100 mg (dry weight) of glycosaminoglycans (expressed as uronic acid); 62.5% of the uronic acids corresponded to hyaluronic acid, the remaining consisted of sulfated glycosaminoglycans (chondroitin-4-(-6) sulfates, and dermatan and heparan sulfates) with different degrees of sulfation.     

Ultrastructure of the milk fat globule membrane with and without triglyceride

Summary The primary milk fat globule membrane (MFGM) around freshly secreted milk fat globules consists of a unit membrane separated from the triglyceride core by a dense material. This dense material may widen to include cytoplasmic organelles or may form small blebs. Preincubation and fixation of the globules at temperatures between 4° C and 60° C has no effect on the width or appearance of the dense material. Isolated MFGM profiles show structures identical to those found on intact globules. The dense material on the isolated MFGM profiles is unaffected by extractions which remove essentially all the triglyceride present in the pellets of MFGM.The structure of the primary MFGM is therefore independent of any triglyceride content and the earlier suggestions that the dark material represented a triglyceride layer of high melting point adsorped during cooling of the globules after milking are not supported by the work described in this paper.     

Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk

Fat globule membranes have been isolated from bovine colostrum and bovine milk by the dispersion of the fat in sucrose solutions at 4 degrees C and fractionation by centrifugation through discontinuous sucrose gradients. The morphology and enzymic characteristics of the separated fractions were examined. Fractions comprising a large proportion of the total extracted membrane were thus obtained having high levels of the Golgi marker enzymes UDP-galactose N-acetylglucosamine beta-4-galactosyltransferase and thiamine pyrophosphatase. A membrane-derived form of the galactosyltransferase has been solubilized from fat and purified to homogeneity. This enzyme is larger in molecular weight than previously studied soluble galactosyltransferases, but resembles in size the galactosyltransferase of lactating mammary Golgi membranes. In contrast, when fat globule membranes were prepared by traditional procedures, which involved washing the fat at higher temperatures, before extraction, galactosyltransferase was not present in the membranes, having been released into supernatant fractions, When the enzyme released by this procedure was partially purified and examined by gel filtration, it was found to be of a degraded form resembling in size the soluble galactosyltransferase of milk. The release is therefore attributed to the action of proteolytic enzymes. Our observations contrast with previous biochemical studies which suggested that Golgi membranes do not contribute to the milk fat globule membrane. They are, however, consistent with electron microscope studies of the fat secretion process, which indicate that secretory vesicle membranes, derived from the Golgi apparatus, may provide a large proportion of the fat globule membrane.     

Differential release of proteins from bovine fat globule membrane

Alkaline phosphatase and 5'-nucleotidase are covalently linked to phosphatidylinositol in bovine fat globule membrane, as demonstrated by their release following treatment with phospholipase C specific for phosphatidylinositol. The failure of this treatment to liberate phosphodiesterase I may indicate that it has a variant linkage resistant to release. In a test of exposure at the membrane surface, alkaline phosphatase and phosphodiesterase I, but not 5'-nucleotidase, were released from fat globule membrane by treatment with proteinase K. These apparent differences in accessibilities of membrane surface proteins suggest that attachment to phosphatidylinositol does not necessarily impart greater exposure to proteins with which it is linked.     

Galactosylceramide is the major cerebroside of human milk fat globule membrane.

Glycolipids were prepared from human milk fat globule membranes and analyzed by t.l.c. on silica gel and borated silica gel plates. Galactosylceramide accounted for 88% of monohexosylceramide and glucosylceramide for only 12%. Altogether they were more abundant than lactosylceramide. Galactosylceramide was shown to contain hydroxy fatty acids but glucosyl and lactosylceramide contained only non-hydroxy fatty acids.