Purification of membrane-bound lactoferrin from the human milk fat globule membrane

Although lactoferrin is known as a basic soluble glycoprotein, the presence of the membrane-bound form of this protein has also been demonstrated in human milk. Membrane-bound lactoferrin was extracted from the human milk fat globule membrane with a detergent mixture of 1% Tween-20, 0.5% C12E8, and 0.5 M KCl in 20 mM Tris-HCl (pH 7.4). Lactoferrin in the detergent-soluble fraction was purified by affinity chromatography with Concanavalin A and by hydrophobic chromatography with phenyl-Superose. The purified protein gave a single band of 80 kDa by SDS-PAGE. Its N-terminal amino acid sequence was consistent with that of human lactoferrin.     

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.     

The butyrophilin (BTN) gene family: from milk fat to the regulation of the immune response

Butyrophilins (BTN) belong to the immunoglobulin (Ig) superfamily of transmembrane proteins. These molecules are of increasing interest to immunologists, as they share a structural homology with B7 family members at the extracellular domain level. Moreover, a role of these molecules has been suggested in the negative regulation of lymphocyte activation for almost all the BTN that have been studied. In addition, the expression of some BTN family members has been reported to be associated with autoimmune diseases. Over the last few years, the number of BTN and BTN-like members has greatly increased. In this study, the butyrophilin family in mammals has been revisited, using phylogenetic analysis to identify all the family members and the phylogenetic relations among them, and to establish a standard nomenclature. Fourteen BTN groups were identified that are not all conserved between mammalian species. In addition, an overview of expression profiles and functional BTN data demonstrates that these molecules represent a new area of investigation for the design of future strategies in the modulation of the immune system.     

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.     

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.     

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.     

Antiserum to the milk fat globule membrane. Preparation and capacity to suppress milk secretion

A procedure is described for preparing rabbit antiserum to goat milk fat globule membrane. This membrane is derived from the secretory surface of the lactating cell. Immunoelectrophoresis and enzyme-linked immunosorbent assay showed that antibody development reached maximal levels in about 6–8 weeks. Infusion of 5–10 ml of this antiserum into the lactating mammary gland of goats via the teat canal depressed milk yields temporarily on the infused side to 60–80% of normal. Ordinary serum from rabbit, goat or human did not evoke such a response and rabbit complement was not essential for the effect. Fractionation showed that the globulin fraction of the antiserum contained the milk-suppressing principle. Milk from the antiserum-infused side of the udder showed extensive and tenacious clumping of fat globules on standing 12–24 h. The inhibition of milk flow by antibodies to the secretory membrane resembles a previously observed inhibition following infusion of concanavalin A or its succinyl derivative. Binding of antibodies or lectins which recognize specific surface protein components of the lactating cell appears to be involved in the suppression mechanism. The possible relevance of our findings to autoimmune suppression of exocytosis is noted.     

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.     

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.     

Use of proteomic methodology for the characterization of human milk fat globular membrane proteins

Characterization of the major human milk fat globular membrane proteins was carried out using proteomic techniques comprising two-dimensional polyacrylamide gel electrophoresis, followed by in situ PNGase F and trypsin digestion. Matrix-assisted laser desorption/ionization quadrupole time-of-flight and electrospray ionization mass spectrometry identified seven major protein components: alpha-lactalbumin, lysozyme precursor, beta-casein, clusterin, lactotransferrin, polymeric immunoglobulin receptor precursor, and human milk fat globule EGF-factor 8 protein. Sequence information on the protein-associated glycans was determined by matrix-assisted laser desorption-ionization quadrupole time-of-flight hybrid mass spectrometry. This glycan analysis revealed interesting fucosylation branching patterns which may be influential in maternal protection of the newborn against bacterial and viral pathogenic attack.     

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.     

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.     

Ultrastructural analysis of milk fat globule membranes of colostrum and human milk

Lacteous fat globules with their membranes from human milk and calostrum were studied by scanning and transmission electron microscopy. The first appear as spheroidal structures with some irregularities on the surface. Under the transmission electron microscope these irregularities are composed of islets from a material morphologically similar to cytoplasm, and with structures that resemble a fragment of rugose endoplasmic reticulum. The membranes in specimens fixed immediately after secretion are tri-layered, similar in appearance to those of a single membrane, but in samples fixed between two and four hours after secretion the details are unclear. Through freeze-etching, the laminar aspect of the fat globules is observed.