Human Milk Mucin Research Articles

Human Milk Mucin 1 and Mucin 4 Inhibit Salmonella enterica Serovar Typhimurium Invasion of Human Intestinal Epithelial Cells In Vitro1–3

Many human milk glycans inhibit pathogen binding to host receptors and their consumption by infants is associated with reduced risk of disease. Salmonella infection is more frequent among infants than among the general population, but the incidence is lower in breast-fed babies, suggesting that human milk could contain components that inhibit Salmonella. This study aimed to test whether human milk per se inhibits Salmonella invasion of human intestinal epithelial cells in vitro and, if so, to identify the milk components responsible for inhibition. Salmonella enterica serovar Typhimurium SL1344 (SL1344) invasion of FHs 74 Int and Caco-2 cells were the models of human intestinal epithelium infection. Internalization of fluorescein-5-isothiocyanate–labeled SL1344 into intestinal cells was measured by flow cytometry to quantify infection. Human milk and its fractions inhibited infection; the inhibitory activity localized to the high molecular weight glycans. Mucin 1 and mucin 4 were isolated to homogeneity. At 150 μg/L, a typical concentration in milk, human milk mucin 1 and mucin 4 inhibited SL1344 invasion of both target cell types. These mucins inhibited SL1344 invasion of epithelial cells in a dose-dependent manner. Thus, mucins may prove useful as a basis for developing novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by Salmonella and related pathogens.

Human milk mucins inhibit salmonella typhimurium invasion of human epithelial cells

Salmonella infection is more frequent among infants than the general population, but the incidence is lower in breastfed babies. This study was to test whether human milk per se inhibited salmonella invasion of human intestinal epithelial cells in vitro, and to identify the milk components likely to be responsible for any inhibition. Salmonella typhimurium SL1344 invasion of FHs 74 Int and CaCo‐2 cells were the models of human intestinal epithelium infection. Internalization of FITC‐labeled salmonella into intestinal cells was measured by FACS analysis to quantify infection. Pooled human milk and its fractions were tested for their ability to inhibit infection, including two human milk mucins, mucin 1 and mucin 4. At 150 ng/mL, approximating the amount in milk, human milk mucin 1 and mucin 4 inhibited salmonella invasion of both cell types. They also bound Salmonella typhimurium SL1344 in a dose‐dependent manner. Thus, mucins may prove useful as a basis for the development of novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by salmonella, and perhaps other pathogens. Supported by NIH HD013021 and AI075563.

Antiviral Activity of Purified Human Breast Milk Mucin

Human breast milk is known to contain numerous biologically active components which protect breast fed infants against microbes, viruses, and toxins. The purpose of this study was to purify and characterize the breast milk mucin and determine its anti-poxvirus activity. In this study human milk mucin, free of contaminant protein and of sufficient quantity for further analysis, was isolated and purified by Sepharose CL-4B gel filtration and cesiumchloride density-gradient centrifugation. Based on the criteria of size and appearance of the bands and their electrophoretic mobility on sodium dodecyl sulfate polyacrylamide-gel electrophoresis, Western blotting together with the amino acid analysis, it is very likely that the human breast milk mucin is MUC1. It was shown that this breast milk mucin inhibits poxvirus activity by 100% using an inhibition assay with a viral concentration of 2.4 million plaque-forming units/ml. As the milk mucin seems to aggregate poxviruses prior to their entry into host cells, it is possible that this mucin may also inhibit other enveloped viruses such as HIV from entry into host cells.

Unique occurrence of the 1CF11 carbohydrate epitope in primate saliva.

We examined a large number of individual human and animal saliva samples for the reactivity with ICF11, a mouse monoclonal antibody previously produced for the characterization of human milk mucin and apparently recognizing a certain carbohydrate antigenic structure shared by various human glycoproteins in secretions. The results obtained here confirm the unique occurrence of ICF11 epitope in each and every saliva sample from humans and Old world monkeys as well, though a vast variety was observed among individual saliva samples in the immunological reactivity with ICF11.

Human Milk Glycoconjugates That Inhibit Pathogens

Breast-fed infants have lower incidence of diarrhea, respiratory disease, and otitis media. The protection by human milk has long been attributed to the presence of secretory IgA. However, human milk contains large numbers and amounts of complex carbohydrates, including glycoproteins, glycolipids, glycosaminoglycans, mucins, and especially oligosaccharides. The oligosaccharides comprise the third most abundant solid constituent of human milk, and contain a myriad of structures. Complex carbohydrate moieties of glycoconjugates and oligosaccharides are synthesized by the many glycosyltransferases in the mammary gland; those with homology to cell surface glycoconjugate pathogen receptors may inhibit pathogen binding, thereby protecting the nursing infant. Several examples are reviewed: A fucosyloligosaccharide inhibits the diarrheagenic effect of stable toxin of Escherichia coli. A different fucosyloligosaccharide inhibits infection by Campylobacter jejuni. Binding of Streptococcus pneumoniae and of enteropathogenic E. coli to their respective receptors is inhibited by human milk oligosaccharides. The 46-kD glycoprotein, lactadherin, inhibits rotavirus binding and infectivity. Low levels of lactadherin in human milk are associated with a higher incidence of symptomatic rotavirus in breast-fed infants. A mannosylated glycopeptide inhibits binding by enterohemorrhagic E. coli. A glycosaminoglycan inhibits binding of gp120 to CD4, the first step in HIV infection. Human milk mucin inhibits binding by S-fimbriated E. coli. The ganglioside, GM1, reduces diarrhea production by cholera toxin and labile toxin of E. coli. The neutral glycosphingolipid, Gb3, binds to Shigatoxin. Thus, many complex carbohydrates of human milk may be novel antipathogenic agents, and the milk glycoconjugates and oligosaccharides may be a major source of protection for breastfeeding infants.

Detection of Large Fragments of the Human Milk Mucin MUC‐1 in Feces of Breast‐Fed Infants

SummaryEvidence is growing that high‐molecular‐weight glycoproteins (mucins) of human milk bind to certain pathogenic microorganisms and thus could interfere with their colonization of the infant gut. This investigation sought to determine whether one of the two principal milk mucins, MUC‐1 (also known as PAS‐0 and episialin), persists into the feces. Fecal samples from seven breast‐fed and seven formula‐fed infants, aged from 20 days to 6 months, were analyzed. Glycoproteins in the specimens, solubilized with sodium dodecylsulfate, mercaptoethanol, and heat, were resolved by gel electrophoresis. The gels were blotted on nitrocellulose sheets that were immunostained with monoclonal antibodies specific for epitopes in the tandem repeat region of MUC‐1. Mucin fragments of ∼200,000 MW were detected in specimens from three of the seven breast‐fed subjects; only one of them showed evidence of the intact mucin. Four breast‐fed and all seven formula‐fed babies were negative for the mucin in their stools. While these results provide evidence of a variable degree of breakdown, we speculate that MUC‐1 resists degradation in the gut and thus may help bind host‐detrimental microorganisms and inhibit their colonizing and infecting actions.

Regulation of sialomucin production in colon carcinoma cells

In this study, we investigated the biochemical basis for the induction of sialomucin production in HT-29 LMM human colon carcinoma cells. Previous studies showed that conditioned medium from organ cultures of normal human colonic connective tissues (NCCM) stimulated the biosynthesis of high molecular weight sialoglycoproteins (M(r) 740,000 and 450,000) by colon carcinoma cells in vitro. Using monoclonal antibodies specific for human milk mucin, we determined that the polypeptide core of these sialomucins was the polymorphic epithelial mucin core peptide coded by the MUC1 gene. Northern analysis showed that cells treated with NCCM expressed a higher level of MUC1 poly(A)+ mRNA than untreated cells. The UDP-GalNAC: polypeptide N-acetylgalactosaminyltransferase activity in microsomal fractions from these cells did not change upon NCCM treatment. The ratios of oligosaccharides with various negative charges secreted by HT-29 LMM cells in the presence of benzyl-alpha-D-N-acetylgalactosaminide were not affected by NCCM treatment indicating that the degree of sialylation of O-linked carbohydrate chains is not influenced by NCCM treatment. [3H]Glucosamine pulse-chase labeling studies using antibodies specific for a COOH-terminal unglycosylated region of the MUC1 mucin core peptides suggested that NCCM treatment of HT-29 LMM cells did not change the rate of MUC1-related sialomucin processing.

Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis.

Acute gastrointestinal infections due to rotaviruses and other enteric pathogens are major causes of morbidity and mortality in infants and young children throughout the world. Breast-feeding can reduce the rate of serious gastroenteritis in infants; however, the degrees of protection offered against rotavirus infection vary in different populations. The mechanisms associated with milk-mediated protection against viral gastroenteritis have not been fully elucidated. We have isolated a macromolecular component of human milk that inhibits the replication of rotaviruses in tissue culture and prevents the development of gastroenteritis in an animal model system. Purification of the component indicates that the antiviral activity is associated with an acidic fraction (pI = 4.0-4.6), which is free of detectable immunoglobulins. Furthermore, high levels of antiviral activity are associated with an affinity-purified complex of human milk mucin. Deglycosylation of the mucin complex results in the loss of antiviral activity. Further purification indicated that rotavirus specifically binds to the milk mucin complex as well as to the 46-kD glycoprotein component of the complex. Binding to the 46-kD component was substantially reduced after chemical hydrolysis of sialic acid. We have documented that human milk mucin can bind to rotavirus and inhibit viral replication in vitro and in vivo. Variations in milk mucin glycoproteins may be associated with different levels of protection against infection with gastrointestinal pathogens.

Reactivity of anti-human milk fat globule antibodies with synthetic peptides.

The nucleotide sequence of partial cDNA clones coding for the core protein of a human polymorphic epithelial mucin has recently been obtained, this mucin consists of a highly conserved 60 bp tandem repeat and the amino acids commonly found are PDTRPAPGSTAPPAHGVTSA. We synthesized three peptides, 1) P1.24 containing the 20 amino acids and four amino acids (PDTR) of the adjoining repeat; 2) P1.15 consisting of the first fifteen (PDTRPAPGSTAPPAH) and P1.09 the second nine amino acids (GVTSAPDTR) of peptide P1.24. The reactivities of the synthetic peptides with mAb known to react with breast cancer (BC1, BC2, BC3, HMFG-1, 3E1.2, and RCC-1) were studied. The synthetic peptide, P1.24, corresponding to the antigenic sequence predicted from the tandem repeat reacted with antibodies BC1, BC2, and BC3 (known to react with human milk mucin and mucin expressed in breast cancer) and the antibody HMFG-1 which was used to select the cDNA clones. In addition, the epitopes recognized by BC1, BC2, and BC3 appear to be in the same region of the molecule represented by their reactions with the nine amino acids in peptide P1.09 (GVTSAPDTR). By contrast, other antibodies such as 3E1.2 which reacts only weakly with components of human milk, and RCC-1 that detects a low Mr component (95 kDa) in breast cancer, had no specific reaction with the synthetic peptides, indicating that their epitopes are distinct from those of BC1, BC2, BC3, and HMFG-1. Inasmuch as the antibodies HMFG-1, BC1, BC2, and BC3 react with the fully processed milk mucin, it is likely that some of the peptide is exposed, even in the fully glycosylated molecule. Identification of the different epitopes could lead to the development of "second generation" mAb with enhanced specificity for breast carcinoma using the appropriate synthetic peptides as immunogens.