Patrice Martin, GABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France
Z. Krupova1,2, C. Pechoux1, C. Henry3, M. Dumarest1, P. Defrenaix2, and P. Martin1
1. GABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France;
2. EXCILONE, Elancourt, France;
3. INRA, UMR MICALIS, Plateforme d’Analyse Protéomique Paris Sud Ouest (PAPPSO),
Université Paris-Saclay, Jouy-en-Josas, France
Introduction: The biosynthesis pathways responsible for the production of milk are known in general outlines but the release and function of extracellular vesicles (EVs) from mammary epithelial cells (MEC) in milk is by far less known. Studying a goat model with a “naturally occurring KO” of the gene (CSN1S1) encoding αs1-casein, we have shown that in goats homozygous for the null allele (O/O), there is a chronic endoplasmic reticulum (ER) stress due to the accumulation of the other caseins in this compartment, slowing down their secretion. Here we report on consequences of the absence of αs1-casein on MEC dysfunction and EVs secretion.
Material and Methods: An improved method based on the combination of successive steps associated to biochemical characteristics of milk components was first developed for purification of milk-derived exosomes. The quality of exosomes was validated morphologically by transmission electron microscopy (TEM) with negative staining (uranyl acetate). The specific exosome protein markers were detected by Western blot and the size distribution was measured by DLS Zetasizer (Malvern) and qNANO (TRPS, Izon). Once validated, their proteome was acquired by LC-MS/MS and nucleic acid content (total RNA and miRNA) by NGS.
Results and Discussion: Our purification method gives milk-derived exosome populations free of contamination by other EVs and milk components, at sufficient concentrations to perform a full characterization while preserving their morphology. Nearly 280 proteins involved in the biogenesis of exosomes and Multi Vesicular Bodies formation, their adhesion and internalization as well as proteins associated with the membrane transport, were identified, among which 18 and 23 proteins differed between αs1-casein “KO” (O/O) and wild-type (A/A) genotypes, respectively. Ongoing profiling of RNA from milk-derived exosomes has already identified over 230 miRNA among which some (miR-148a-3p, miR30a-5p, miR-200a and miR-200b) are highly represented whatever the genotype, whereas miR-21-5p seems to be in less amount in AA goats. The presence of mRNA encoding major milk proteins (α-lactalbumin and αs2-casein) in these vesicles confirm their MEC origin.
Conclusions: Our results demonstrate the effectiveness and the potential of our method to purify milk-derived exosomes and confirm the impact of a deficit in αs1-casein on the MEC secretion phenotype, thus providing evidences that αs1-casein genotype is capable to modulate the repertoire of molecules conveyed by exosomes which is of crucial interest since miRNA stable in extracellular vesicles may play an important regulatory role in the young and may account for the health benefits of milk for the consumer.
Acknowledgements: This study was funded by the National Agency for Research (MilkChEST, ANR-12-BSV6-0013-04) and by the GIS APIS-GENE. We thank Claudia Bevilacqua (Microgenomics platform from the @BRIDGe core facility) for her invaluable assistance regarding qPCR.Download PDF