A major prolactin-binding complex on human milk fat globule membranes contains cyclophilins A and B: the complex is not the prolactin receptor

Prolactin (PRL) in milk influences maturation of gastrointestinal epithelium and development of both the hypothalamo-pituitary and immune systems of offspring. Here, we demonstrate that most PRL in human milk is part of a novel, high-affinity, multicomponent binding complex found on the milk fat globule membrane and not in whey. To examine properties of the complex, a sensitive ELISA was developed such that human PRL (hPRL) binding to the complex was measured by loss of hPRL detectability; thus, as much as 50 ng of hPRL was undetectable in the presence of 10 μl of human milk. Using the same methodology, no comparable complex formation was observed with human serum or amniotic fluid. hPRL complexation in milk was rapid, time dependent, and cooperative. Antibodies to or competitors of the hPRL receptor (placental lactogen and growth hormone) showed the hPRL receptor was not involved in the complex. However, hPRL complexation was antagonized by cyclosporine A and anti-cyclophilins. The complex was very stable, resisting dissociation in SDS, urea, and dithiothreitol. Western analysis revealed an ～75-kDa complex that included hPRL, cyclophilins A and B, and a 16-kDa cyclophilin A. Compared with noncomplexed hPRL, complexed hPRL in whole milk showed similar activation of STAT5 but markedly delayed activation of ERK. Alteration of signaling suggests that complex formation may alter hPRL biological activity. This is the first report of a unique, multicomponent, high-capacity milk fat reservoir of hPRL; all other analyses of milk PRL have utilized defatted milk.     

Presence and genetic polymorphism of an epithelial mucin in milk of the goat (Capra hircus).

1. Analysis of individual samples of goat's milk by SDS-PAGE confirmed that they contain a polymorphic, high molecular weight (M(r) greater than 205 kDa) glycoprotein. 2. On SDS-gels, the polymorphism takes the form of two bands of variable mobility which usually stain with equal intensity. This polymorphism resembles that detected in milk mucins of other species and is best explained by an expression of codominant genes containing variable numbers of a tandemly repeated 60-base segment. 3. Analysis of milk fractions provided evidence that the goat mucin is exclusively a membrane protein, and that it can be purified from other fat globule proteins by gel filtration and peanut lectin affinity chromatography. 4. Among proteins in the goat milk fat globule, the mucin appears to be a strong immunogen but the resulting antibodies applied to Western blots only stained the cow's milk mucin mildly and the guinea pig and human milk mucins not at all.     

Proteome profile and biological activity of caprine, bovine and human milk fat globules

Upon combining bidimensional electrophoresis with monodimensional separation, a more comprehensive analysis of the milk fat globule membrane has been obtained. The proteomic profile of caprine milk fat globules revealed the presence of butyrophilin, lactadherin and perilipin as the major proteins, they were also associated to bovine and human milk fat globule membranes. Xanthine dehydrogenase/oxidase has been detected only in monodimensional gels. Biological activity of milk fat globules has been evaluated in Caco2-cells, as a representative model of the intestinal barrier. The increase of cell viability was indicative of a potential nutraceutical role for the whole milk fat globule, suggesting a possible employment in milk formula preparation.     

Galactose transfer in the membranes of human milk fat globules (author's transl)]

Galactosyltransferase which catalyzes the transfer from UDP-galactose to either endogeneous glycoproteins, free N-acetylglucosamine or N-acetylglucosaminyl residues in the carbohydrate portion of glycoproteins, or to glucose when alpha-lactalbumin is added, occurs in human milk fat globule membranes. Various treatments (washing of membranes, freezing and thawing) did not affect this activity. In the presence of Triton X-100, the enzyme shows appreciable latency, This detergent was then used to solubilize the enzyme and to study its main characteristics. A competition and a heat stability experiment show that only one enzyme acts on two substrates (free N-acetylglucosamine or desialyzed and degalactosylated fetuin). UDP-galactose hydrolase activities were very low compared to those of the bovine milk fat globule membranes. Other characteristic enzymes of Golgi vesicles were found in human milk fat globules membranes. It is of interest to find out whether this is the result of contamination with cytoplasmic particles or whether it reflects the participation of Golgi vesicles in human milk fat globule secretion.     

Gel electrophoretic analysis of proteins in human milk and colostrum

Protein patterns of skim milk and fat globule phases were relatively unchanging throughout 9 months lactation, the only notable differences being a rise in lysozyme and a decline up to 6 months with increase thereafter in lactoferrin. Profound changes in protein patterns of the breast secretion were observed during the first week postpartum. True colostrum, in which peptides of sIgA dominate the protein patterns, exists for at most the 2 initial days of secretion. Proteins of mature milk are not coordinated to appear in the colostral secretion simultaneously. A band corresponding to alpha-lactalbumin is present from the initial secretion; that for beta-casein emerges approximately 2 days layer.     

PAS IV, an integral membrane protein of mammary epithelial cells, is related to platelet and endothelial cell CD36 (GP IV).

PAS IV is a 78-kDa (bovine) to 80-kDa (human) integral membrane glycoprotein of unknown function which is found in mammary epithelial cells. We now report the purification of human PAS IV and native bovine PAS IV from the milk fat globule membrane (MFGM), a preparation of apical plasmalemma from epithelial cells of lactating mammary tissue. N-Terminal sequence analyses of human and bovine PAS IV revealed homology to the N-terminal sequence of the 88-kDa human endothelial and platelet glycoprotein CD36. The similarity of MFGM PAS IV to platelet CD36 was further established by immunoblots of purified platelet CD36 and MFGM PAS IV with MFGM PAS IV specific antiserum. The removal of N-linked oligosaccharides from PAS IV and CD36 by treatment with endoglycosidase F reduced the apparent Mr of both proteins to approximately 57,000. These data suggest that PAS IV and CD36 are similar if not identical polypeptides that undergo cell type specific glycosylation.     

The Bovine Lactophorin C-Terminal Fragment and PAS6/7 Were Both Potent in the Inhibition of Human Rotavirus Replication in Cultured Epithelial Cells and the Prevention of Experimental Gastroenteritis

Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. We have found that high-M_r glycoprotein fraction (F1) from cow’s milk whey has potent inhibitory activity against human rotavirus (HRV) in cell culture. The present study was undertaken to identify and characterize the components responsible for this inhibitory activity. F1 was initially heated at 95 °C for 30 min, rendering milk antibodies inert, subjected to ammonium sulfate fractionation, and then resolved by two-dimensional polyacrylamide gel electrophoresis. After electroelution, we found that a heat-stable milk protein lactophorin C-terminal fragment (LP16) and bovine milk fat globule membrane protein PAS6/7 strongly inhibited the replication of HRV MO strains in MA104 cells. Furthermore, we found that prophylactic oral administration of F1 once before inoculation of the HRV MO strain obviously prevented the development of diarrhea in vivo. These non-immunoglobulin components are a promising candidate for a prophylactic food additive against HRV infection.     

Regulation and functional relevance of milk fat globules and their components in the mammary gland

Mammary gland and epithelial cells are unique to mammals and are under the control of lactogenic hormones such as prolactin. Recent findings indicated that major components of milk fat globule membrane (MFGM) are under the control of lactogenic hormones, and that the major components butyrophilin and xanthine oxidoreductase are indispensable for milk fat secretion. Further, prolactin signaling is negatively controlled by two highly related protein tyrosine phosphatases, PTP1B and TC-PTP. Milk fat globule EGF factor 8 (MFG-E8) is one of the major components of MFGM and is upregulated during lactation. MFG-E8 is further upregulated in the involuting mammary gland. MFG-E8 on exosome-like membrane vesicles in the milk recovered from post-weaning but not lactating mammary glands exhibits higher binding activity to phosphatidylserine and apoptotic mammary epithelial cells, and serves as a link between apoptotic mammary epithelial cells and phagocytes. Recent reports using MFG-E8 deficient mice support the view that MFG-E8 is indispensable for eliminating apoptotic mammary epithelial cells during involution.     

Transfert du galactose dans les membranes des globules lipidiques du lait humain

Galactose transfer in the membranes of human milk fat globulesGalactosyltransferase which catalyzes the transfer from UDP-galactose to either endogeneous glycoproteins, free N-actylglucosamine or N-acetylglucosaminyl residues in the carbohydrate portion of glycoproteins, or to glucose when α-lactalbumin is added, occurs in human milk fat globule membranes. Various treatments (washing of membranes, freezing and thawing) did not affect this activity. In the presence of Triton X-100, the enzyme shows appreciable latency. This detergent was then used to solubilize the enzyme and to study its main characteristics. A competition and a heat stability experiment show that only one enzyme acts on two substrates (free N-acetylglucosamine or desialyzed and degalactosylated fetuin). UDP-galactose hydrolase activities were very low compared to those of the bovine milk fat globule membranes. Other characteristic enzymes of Golgi vesicles were found in human milk fat globules membranes. It is of interest to find out whether this is the result of contamination with cytoplasmic particles or whether it reflects the participation of Golgi vesicles in human milk fat globule secretion.     

Evaluation of the anti-adhesive effect of milk fat globule membrane glycoproteins on Helicobacter pylori in the human NCI-N87 cell line and C57BL/6 mouse model

Background: The interest in non‐antibiotic therapies for Helicobacter pylori infections in man has considerably grown because increasing numbers of antibiotic‐resistant strains are being reported. Intervention at the stage of bacterial attachment to the gastric mucosa could be an approach to improve the control/eradication rate of this infection. Materials and Methods: Fractions of purified milk fat globule membrane glycoproteins were tested in vitro for their cytotoxic and direct antibacterial effect. The anti‐adhesive effect on H. pylori was determined first in a cell model using the mucus‐producing gastric epithelial cell line NCI‐N87 and next in the C57BL/6 mouse model after dosing at 400 mg/kg protein once or twice daily from day ?2 to day 4 post‐infection. Bacterial loads were determined by using quantitative real‐time PCR and the standard plate count method. Results: The milk fat globule membrane fractions did not show in vitro cytotoxicity, and a marginal antibacterial effect was demonstrated for defatted milk fat globule membrane at 256 μg/mL. In the anti‐adhesion assay, the results varied from 56.0 ± 5.3% inhibition for 0.3% crude milk fat globule membrane to 79.3 ± 3.5% for defatted milk fat globule membrane. Quite surprisingly, in vivo administration of the same milk fat globule membrane fractions did not confirm the anti‐adhesive effects and even caused an increase in bacterial load in the stomach. Conclusions: The promising anti‐adhesion in vitro results could not be confirmed in the mouse model, even after the highest attainable exposure. It is concluded that raw or defatted milk fat globule membrane fractions do not have any prophylactic or therapeutic potential against Helicobacter infection.     

Milk fat globule membranes devoid of intramembranous particles

When isolated milk fat globule membranes from bovine, human, and murine (rat) milk were examined by freeze-fracturing most of the membrane faces were devoid of membrane-intercalated particles whereas a minor portion showed relatively few particles, either in clusters or in apparent random distribution. A reduced particle density was also noted in membranes of intra-alveolar milk fat globules of cows and rats, in contrast to high particle densities in the apical plasma membrane of lactating epithelial cells. The observations suggest that certain membrane constituents recognized as intramembranous particles either are displaced from the region of the apical surface of the mammary epithelial cell which is involved in milk fat globule budding or are dislocated and rearranged during the budding process.     

Differential scanning calorimetry of milk fat globule membranes

Differential scanning calorimetry was employed as an aid in examining the structure of the bovine milk fat globule membrane. At least six major endotherms are observed between 10 and 90°C, corresponding to order-disorder transitions of discrete structural domains of the membrane. These endothermic transitions occur at 16, 28, 43, 58, 68, and 75°C. The transitions occurring between 10 and 50°C were reversible, suggesting the involvement of lipid. However, the high temperature transitions were irreversible. The calorimetric C transition, centered at 43°C, was shown to involve neutral lipid, since the endotherm was reversible, insensitive to proteolysis, and similar to the endotherm of the isolated neutral lipid fraction of the milk fat globule membrane. The glycolipid and phospholipid fractions of the milk fat globule membrane yielded endotherms outside of the temperature range of the C transition. Another endotherm, the D transition (58°C), was found to involve the denaturation of the major membrane coat protein, butyrophilin (band 12). Evidence for this assignment included the following observations: (i) the nearly selective proteolysis of butyrophilin resulted in the complete removal of the D transition, (ii) the butyrophilin-enriched, Triton X-100-insoluble pellet of milk fat globule membrane yielded a relatively normal D transition, and (iii) the irreversible, disulfide-stabilized aggregation of butyrophilin occurred in the membrane solely at the temperature of the D transition. Furthermore, no other prominent milk fat globule membrane polypeptide formed these non-native disulfide crossbridges during the D transition. The sources of the other major endotherms of the milk fat globule membrane have not yet been assigned.     

Proteomic characterization of human milk fat globule membrane proteins during a 12 month lactation period

The milk fat globule membrane (MFGM) contains proteins which have been implicated in a variety of health benefits. Milk fat globule membrane proteins were isolated from human milk during a 12 month lactation period and subjected to in-solution digestion and liquid chromatography tandem mass spectrometry analysis. Data were pooled, and our results showed that 191 proteins were identified. Relative quantification of the identified MFGM proteins during the course of lactation was performed by label free spectral counting and differentiation expression analysis, which showed some proteins decreasing during the course of lactation whereas some increased or remained at a relatively constant level. The human MFGM proteins are distributed between intracellular, extracellular, and membrane-associated proteins, and they are mainly involved in cell communication and signal transduction, immune function, metabolism and energy production. This study provides more insights into the dynamic composition of human MFGM proteins, which in turn will enhance our understanding of the physiological significance of MFGM proteins.     

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRL-HMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.     

A 46-kDa antigen associated with estrogen receptor in human breast cancer

A 65-kDa estrogen receptor (ER) protein has been demonstrated both by sucrose gradient analysis and by immunoblot, using anti-ER monoclonal antibodies (MAbs). Since the ER is denatured in many experimental situations, such as formaldehyde fixing of samples for histochemistry and electroimmunoblotting studies, in this work we used a denatured 60-70-kDa ER-rich protein preparation as antigen for mice immunization in order to raise anti-ER MAbs. That material was obtained by affinity purification on an allyl-estradiol matrix of the MCF-7 cytosolic ER, followed by further isolation and enrichment by PAGE. NS-1 myeloma cells and spleen lymphocytes from the immunized mice were fused, and resultant hybridoma colonies were screened by [125I]-estradiol-labelled nuclear ER immunoprecipitation. The isolated MAb, E476, shows a moderate ability to precipitate ER and reacts strongly with a 46-kDa antigen in Western blot assay. The 46-kDa antigen was not detectable in native cytosol but became reactive after 50% ammonium sulfate precipitation of cytosolic proteins. The 46-kDa antigen appeared concentrated in the NaSCN plus estradiol eluate of the affinity column used for cytosolic ER purification. Freshly prepared 60-70-kDa material from the preparative gel electrophoresis did not show any E476 reactivity. However, when the 60-70-kDa proteins were frozen, thawed and speed vacuum concentrated, the 46-kDa antigen became detectable. Storage increased the reactivity of the 60-70-kDa material with the E476 MAb. The 46-kDa antigen was present only in the ER positive cell lines, and was absent in all negative cell lines tested. The 46-kDa protein is also present in the ER positive human breast cancer specimens. We conclude that the 46-kDa protein identified with the E476 MAb in human breast cancer is probably a naturally occurring ER fragment.     

MFG—E8的生物学功能及应用

乳脂球表皮生长因子8（milk fat globuleEGF factorⅧ,MFG—E8）是一种乳汁脂肪小球表面的亲脂性糖蛋白,广泛参与多种细胞问的相互作用,如介导精子与卵子外膜问的结合、维持附睾上皮细胞与精子细胞膜的黏附、参与肠上皮细胞的维持与修复、促进乳腺支化形成和成人组织新生血管形成、增强树突状细胞外泌体功能以及参与吞噬清除凋亡细胞的过程。此外,MFG—E8也显示良好的应用前景,有可能作为潜在的治疗和检测手段。     

BIOCHEMICAL AND MORPHOLOGICAL COMPARISON OF PLASMA MEMBRANE AND MILK FAT GLOBULE MEMBRANE FROM BOVINE MAMMARY GLAND

Purified plasma membrane fractions from lactating bovine mammary glands and membranes of milk fat globules from the same source were similar in distribution and fatty acid composition of phospholipids. The sphingomyelin content of the phospholipid fraction of both membranes was higher than in these fractions from other cell components, β-carotene, a constituent characteristic of milk fat, was present in the lipid fraction of the plasma membrane. Cholesterol esters of plasma membrane were similar in fatty acid composition to those of milk fat globule membranes. Disc electrophoresis of either membrane preparation on polyacrylamide gels revealed a single major protein component characteristic of plasma membrane from other sources. Distinct morphological differences between plasma membrane and milk fat globule membranes were observed in both thin sections and in negatively stained material. Plasma membrane was vesicular in appearance while milk fat globule membranes had a platelike aspect. These observations are consistent with derivation of fat globule membrane from plasma membrane accompanied by structural rearrangement of membrane constituents.     

Release of remnant plasma membrane from milk fat globules by Triton X-100

The nonionic detergent, Triton X-100, was investigated as an agent for releasing plasma membrane from milk fat globules. The sedimentable material ($\text{50 000 × g}$, 1 h) derived by treating washed goat globules with the detergent (0.2%) was compared to membrane made by the classical globule churning procedure. Characterization included lipid and protein analyses, gel electrophoresis of peptide components, determination of enzymatic activities, and examination with the electron microscope. The results established that the detergent-released material is membrane with similarities to the product by churning. Evaluation of variables revealed that a detergent concentration of 0.1 to 0.2% and reaction temperature of 20–22°C appear optimum with respect to membrane yield when a reaction time of 2 min is employed. At higher detergent concentrations or temperatures removal of phospholipid from the membrane was maximized. Triton X-100 was observed to release membrane from milk fat globules of the goat, human and cow, the latter with a minor procedural modification. The detergent based method is a convenient procedure for obtaining plasma membrane material in good yield for biochemical studies. It also should aid investigations of milk fat globule structure.     

MCF—7细胞在亚多层中细胞膜极性的变化

Sub-multilayer of MCF-7 cell, an established human breast carcinoma cell line, was achieved by culturing the cells on millipore filters for a long time. The superficial layer cells maintained their membrane polarity features as MCF-7 cells in monolayers did. MAM-6, a human milk fat globule membrane antigen, was polarized distributed in apical domain of 97.5% superficial layer cells revealed by immunoperoxidase cytochemistry. Whereas, among the low layer cells, which had no free surface (apical domain) toward the culture medium and did not show morphological polarity features, only 12.9% expressed surface MAM-6 with weak immunoperoxidase staining and random distribution. But the immunostaining for detecting cytoplasmic MAM-6 in low layer cells was stronger than that in superficial layer cells, indicating that the vectorial delivery and insert of MAM-6 carrying glycoprotein to the plasma membrane seemed to be stopped or declined and became undirectional in the later situation. The study demonstrates that an asymmetric spatial environment, which is composed of a liquid phase space and a solid phase space, is crucial for the establishment of epithelial membrane polarity of MCF-7 cells.     

Sustained co-cultivation with human placenta-derived MSCs enhances ALK5/Smad3 signaling in human breast epithelial cells,leading to EMT and differentiation

The interaction between mammary epithelial cells and their surrounding microenvironment are important in the development of the mammary gland. Thus, mesenchymal stem cells (MSCs), which retain pluripotency for various mesenchymal lineages, may provide a permissive environment for the morphologic alteration and differentiation of mammary epithelial cells. To this end, we investigated whether the interactions between mammary epithelial cells and human placenta-derived MSCs (hPMSC) affect the morphology, proliferation, and differentiation of epithelial cells in a co-culture system. We show that after co-culture with hPMSCs, human mammary epithelial cell lines (MCF-10F and HEMC) underwent significant morphologic alterations and a dramatic increase in ductal–alveolar branching, which was accompanied by a decrease or loss of the epithelial marker E-cadherin and a gain of the mesenchymal markers, α-SMA and vimentin. MCF-10F and HEMC proliferation was also inhibited in the presence of hPMSCs, and this retardation in growth was due to cell cycle arrest. Furthermore, in MCF-10F and HMEC cells, hPMSCs induced the production of lipid droplets, milk fat globule protein, and milk protein lactoferrin, which are markers of functional mammary differentiation. We also noticed an elevation in ALK5 and phosphorylated Smad3 protein levels upon hPMSC co-culture. Strikingly, the changes in morphology, proliferation, and differentiation were reversed by treatment with ALK5 or Smad3 knockdown in MCF-10F/hPMSC co-cultures. Collectively, our findings suggest that co-cultivation with hPMSCs leads to epithelial to mesenchymal transition (EMT) and differentiation of human breast epithelial cells through the ALK5/Smad3 signaling pathway.