Galactose and Sialo-galactose Modifications in Glycoproteins on the Surface of Bovine Milk Exosome Are Essential for Exosome Uptake in Non-bovine Species (OR34-07-19)

Abstract

Objectives Exosomes are natural nanoparticles that can be found in most body fluids including milk. Bovine milk exosomes (BME) are bioavailable and transfer cargos such as RNAs, lipids and proteins across species boundaries. Previous competitor studies implicated galactose-modifications in exosome surface proteins in exosome transport. The objectiveof our study was to assess the importance of glycan modifications in BME surface glycoproteins for BME uptake and distribution in human intestinal cells (FHs cells) and mice. Methods BME were isolated from milk by differential centrifugation (Fig. 1). Putative glycan binding sites on the surfaces of BME were identified by LC/MS-MS and bioinformatics protocols and confirmed by eastern blotting. Surface glycans were altered using proteases, glycosidases and blocking of glycans with lectins for subsequent transport and distribution studies (Table 1). The uptake of BME by FHs cells was assessed using membrane and RNA cargo-labeled BME. The distribution of BME in mice was assessed using BME transfected with fluorophore (IRDye)-labeled synthetic miR-320a in C57BL/6 mice and MGAT-1 tamoxifen inducible conditional knockout (CKO) mice ages 5–8 weeks. One-way ANOVA and Bonferroni's multiple comparison were used for statistical analyses; P < 0.05 was considered significant. Results We identified 49 (N), 13 (O) and 13 (C) putative glycan binding sites on the BME surface, and confirmed the presence of mannose, galactose, N-acetylgalactose, fucose and neuraminate (Fig. 2). Galactose and N-acetylgalactosamine residues on the surface of BME were of particular importance for their uptake by FHs cells, whereas N-acetylglucosamine on the surface of FHs cells was more important than other glycans for BME uptake (Fig. 3). Enzymatic and genetic removal of glycans in BME and MGAT-1 CKO, respectively, caused a loss of BME accumulation in murine livers and pancreas (Fig. 4). Conclusions Distinct glycan features are essential for the uptake of BME in mice. The study laid a foundation to assess the importance of glycan modifications in infant formulas and the homing of drug-loaded exosomes to sites of disease. Funding Sources NIFA, NIH, Bill & Melinda Gates Foundation, PureTech Health, and USDA Hatch & Multistate. J.Z. is a consultant for PureTech. Supporting Tables, Images and/or Graphs

Beneficial Effects of Bovine Milk Exosomes in Metabolic Interorgan Cross-Talk

“…Furthermore, exosomes have been shown to confer protection against the adverse conditions present in the digestive tract, which suggests that the oral intake of these compounds facilitates the availability of their cargos [ 86 , 87 , 88 ]. Besides protecting their cargos, glycoproteins displayed in milk exosomes facilitate their recognition and uptake by the target cell receptors [ 70 , 89 ].…”

Comprehensive Analysis of the Glycome and Glycoproteome of Bovine Milk-Derived Exosomes

“…Multiple glycoproteins located on the surface of exosomes were examined, and their glycosylation profiles greatly affected the interactions of the exosomes with the acceptor cells . A previous report demonstrated that galactose and sialo-galactose modifications of glycoproteins on the surface of BMDEs facilitated the uptake of exosomes by nonbovine species . In contrast, the existence of mannose, N -acetylglucosamine, or lactose residues was reported to inhibit dendritic cells and macrophage internalization of exosomes. , …”

Extracellular Vesicles as an Efficient and Versatile System for Drug Delivery

“…A study on bovine milk-derived EV uptake found that the glycans on membrane proteins of EVs determine their absorption on human intestinal cells and in mouse models [ 120 ]. Among the studied residues, galactose and N -acetylgalactosamine residues on EV proteins and N -acetylglucosamine residues on proteins of intestinal cells were important for EV uptake, as the removal of these glycans by enzymes or by knockout genes completely blocked EV uptake through the intestine and EV accumulation in the liver and pancreas [ 120 ]. EVs that had undergone desialylation were in general taken up more readily compared to normal EVs, but the increase varied between cell lines [ 121 ].…”