The protective effects of human milk-derived peptides on the pancreatic islet biology.

Several epidemiological studies support the protective role of breastfeeding in reducing the risk for type 1 diabetes. Human breast milk is the perfect nutrition for infants and contains many complex proteins, lipids and carbohydrates. In this study, we examined the physiological effects of human milk-derived opioid peptides, ß-casomorphins (BCM), and compared them with bovine-milk-derived opioid peptides on pancreatic hormone regulation and ß-cell regeneration. Exposure of wild-type zebrafish embryos to 50 µg/ml of human BCM-5 and -7 from 3 days post fertilisation until 6 days post fertilisation resulted in an increased insulin domain of expression while exposure to bovine BCM-5 and -7 significantly reduced the insulin domain of expression as analysed by whole-mount in situ hybridisation. These changes may be accounted for by reduced insulin expression or ß-cell number and were mitigated by the µ-opioid receptor antagonist, naloxone. The effect of BCM on ß-cell regeneration was assessed following ablation of ß-cells in Tg (ins: CFP-NTR) zebrafish from 3 days post fertilisation to 4 days post fertilisation, followed by exposure of bovine and human BCM-5 and -7 (50 µg/ml) from 4 days post fertilisation until 7 days post fertilisation. The regenerative capacity of ß-cells was not impeded following exposure to human BCM-5 and -7, whereas the capacity of ß-cells to regenerate following bovine BCM-5 and -7 exposure was reduced. Our data suggest that human BCM-5 and -7 may promote ß-cell development and enable the regeneration of ß-cells, while the bovine-milk-derived peptides, BCM-5 and -7, play an opposite role. These data may provide some biological explanation for the protective effect of breastfeeding on the development of type 1 diabetes.

In vivo endogenous proteolysis yielding beta-casein derived bioactive beta-casomorphin peptides in human breast milk for infant nutrition.

Objective Beta-casein is a major protein in breast milk and an important source for several bioactive peptides that are encrypted within the sequence. Beta-casomorphins (BCMs) are short-chain proteolytic peptides that are derived from the beta-casein protein and have opioid effects in newborns. Human milk is known to contain naturally occurring milk-protein-derived bioactive peptides but the identification of naturally occurring beta-casein-derived BCMs in human breast milk has been limited due to difficulties in the detection of BCM peptides, which are small and circulate in low concentrations. Methods The present study aimed to identify the naturally occurring BCM peptides from beta-casein in human breast milk using liquid chromatography-tandem mass spectrometry. The BCM peptides identified in the breast milk were analysed to predict the milk proteases responsible for the cleavage patterns using a computational tool EnzymePredictor. Results In-depth peptidomics analysis of breast milk samples that were collected at different lactation stages during human lactation revealed the presence of BCMs including BCM-8, -9, -10, and -11 as well as precursors and truncated forms of the original peptide, which suggests that milk protease activity in the mammary gland generates biologically relevant BCMs. Conclusions To our knowledge, this is the first report to describe the presence of naturally occurring human BCM-10 and -11 in breast milk. Our study provides evidence of beta-casein-derived BCM peptides in human milk before infant digestion. Proteases that are present in milk are likely specific in their proteolysis of beta-casein. The identified bioactive BCM-8, -9, -10, and -11 as well as the precursor peptides meet the structural requirements to elicit opioid, immunomodulatory, antioxidative, and satiety functions in newborns.

Dietary Cows' Milk Protein A1 Beta-Casein Increases the Incidence of T1D in NOD Mice.

The contribution of cows' milk containing beta-casein protein A1 variant to the development of type 1 diabetes (T1D) has been controversial for decades. Despite epidemiological data demonstrating a relationship between A1 beta-casein consumption and T1D incidence, direct evidence is limited. We demonstrate that early life exposure to A1 beta-casein through the diet can modify progression to diabetes in non-obese diabetic (NOD) mice, with the effect apparent in later generations. Adult NOD mice from the F0 generation and all subsequent generations (F1 to F4) were fed either A1 or A2 beta-casein supplemented diets. Diabetes incidence in F0⁻F2 generations was similar in both cohorts of mice. However, diabetes incidence doubled in the F3 generation NOD mice fed an A1 beta-casein supplemented diet. In F4 NOD mice, subclinical insulitis and altered glucose handling was evident as early as 10 weeks of age in A1 fed mice only. A significant decrease in the proportion of non-conventional regulatory T cell subset defined as CD4⁺CD25-FoxP3⁺ was evident in the F4 generation of A1 fed mice. This feeding intervention study demonstrates that dietary A1 beta-casein may affect glucose homeostasis and T1D progression, although this effect takes generations to manifest.

Structural characterization of a novel monotreme-specific protein with antimicrobial activity from the milk of the platypus.

Monotreme lactation protein (MLP) is a recently identified protein with antimicrobial activity. It is present in the milk of monotremes and is unique to this lineage. To characterize MLP and to gain insight into the potential role of this protein in the evolution of lactation, the crystal structure of duck-billed platypus (Ornithorhynchus anatinus) MLP was determined at 1.82 Šresolution. This is the first structure to be reported for this novel, mammalian antibacterial protein. MLP was expressed as a FLAG epitope-tagged protein in mammalian cells and crystallized readily, with at least three space groups being observed (P1, C2 and P21). A 1.82 Šresolution native data set was collected from a crystal in space group P1, with unit-cell parameters a = 51.2, b = 59.7, c = 63.1 Å, α = 80.15, ß = 82.98, γ = 89.27°. The structure was solved by SAD phasing using a protein crystal derivatized with mercury in space group C2, with unit-cell parameters a = 92.7, b = 73.2, c = 56.5 Å, ß = 90.28°. MLP comprises a monomer of 12 helices and two short ß-strands, with much of the N-terminus composed of loop regions. The crystal structure of MLP reveals no three-dimensional similarity to any known structures and reveals a heretofore unseen fold, supporting the idea that monotremes may be a rich source for the identification of novel proteins. It is hypothesized that MLP in monotreme milk has evolved to specifically support the unusual lactation strategy of this lineage and may have played a central role in the evolution of these mammals.

Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes.

Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians.

Monotreme lactation protein is highly expressed in monotreme milk and provides antimicrobial protection.

Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female's abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples.