Human milk-derived EV are a major macromolecular component in breast milk with distinct bioactive properties

Bernd Stahl, Nutricia Research Centre for Specialized Nutrition, The Netherlands

Martijn J.C. van Herwijnen1, Marijke I. Zonneveld1,2, Soenita Goerdayal3,
Esther N.M. Nolte–’t Hoen1, Johan Garssen2,4, Bernd Stahl4, A.F. Maarten Altelaar3,
Frank A. Redegeld2, and Marca H.M. Wauben1
1. Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands;
2. Division of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, The Netherlands;
3. Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences;
4. Nutricia Research Centre for Specialized Nutrition, Utrecht, The Netherlands

Human milk is a complex biological fluid containing various macromolecular components with distinctive functions: it provides nutrition, it boosts immunity, it induces tolerance and it supports the epithelial barrier. Generally, each function can be allocated to specific macromolecular structures. The milk fat globules provide most of the lipids while casein micelles provide most of the proteins, making them the nutritional components. The bioactive components of milk are found in the whey fraction, which include antimicrobials, IgA, cytokines and growth factors. In addition, human milk is rich in oligosaccharides that feed the gut microbiota of the newborn. Recently, milk-derived extra-cellular vesicles (EV) have been identified adding another component to milk. However, their importance and function has not been addressed in detail. This in part is due to the difficulty to obtain human milk and to purify EV from other milk components. Consequently, the molecular composition of human milk-derived EV has not been fully elucidated.

In this study, an extensive LC-MS/MS proteomic analysis was performed on EV isolated via
density-based separation from human milk of 7 individual donors. The proteome of milk-derived EV was analyzed for EV-associated markers and compared to the whole milk proteome, which was manually constructed from 38 previously published milk proteomics studies. These included the analysis of whole milk, serum, whey, casein and milk fat globules with a total of 2698 individual proteins.

A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5 and Flotillin-1. Next, the milk-derived EV proteome was compared to the whole milk proteome. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in milk to date. Interestingly, these novel proteins included proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, while milk proteins not associated to EV were involved in protein metabolism. Using site of expression analysis, we identified that immune cells are the likely producers of milk-derived EV rather than mammary epithelial cells.

In conclusion, these data show that milk-derived EV have an extensive proteome and harbor proteins previously unidentified in milk. These novel proteins have the capacity to support the infant’s developing gastrointestinal tract and its immune system. In addition, this study provides an expansion to the whole milk proteome, illustrating that milk-derived EV are a distinct macromolecular component with a unique functional proteome.

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