Identification of Glycoproteins on the Surface of Bovine Milk Exosomes and Intestinal Cells that Facilitate Exosome Uptake in Human Colon Carcinoma Caco‐2 Cells

Abstract

BackgroundExosomes are small, cargo‐containing vesicles that are secreted by donor cells to elicit changes in gene expression and metabolism in recipient cells. In previous studies, we provided evidence that, in addition to endogenous synthesis, exosomes may also be obtained from dietary sources such as bovine milk, and intestinal transport is facilitated by endocytosis.HypothesisGlycoproteins on the surface of bovine milk exosomes and human intestinal cells play essential roles in exosome transport.MethodsExosomes were isolated from bovine milk by differential centrifugation and authenticated as recommended by International Society for Extracellular Vesicles (Fig. 1). Surface proteins or glycan modifications in exosomes or Caco‐2 cells were removed using proteases (Glu‐C, trypsin, Arg‐C, Asp‐N, or proteinase K) or glycosidases [N‐glycosidase F (PNGase F), galactosidase, O‐glycosidase, neuraminidase, N‐acetyl glucosaminidase, or a combination of all]. Controls were incubated with solvent. Exosomes were labeled with FM4–64, and unlabeled fluorophore was removed. Surface peptides released after the treatment were identified using LC/MS‐MS, Mascot and Sequest databases. Glycoproteins on the external surface were identified using a series of tools based on neuronal networks including NetNglyc, NetOglyc, NetCglyc 1.0 and GlycoEP. Transport kinetics were modelled using the Michaelis Menten equation. One‐way ANOVA and Bonferroni's multiple comparison were used for statistical analyses.ResultsWhen exosomes or cells were treated with individual proteases (Fig. 2) or glycosidases (Fig. 3), exosome uptake decreased up to 80% when compared to controls even at prolonged incubation times. Treatment with galactosidase, PNGase F and O‐glycosidase had a stronger effect than other glycosidases. When exosomes or cells were treated with the glycosidase mixture, no uptake was detected or uptake was decreased by 80%, respectively, in comparison to controls (Fig. 4). 149 total proteins were identified in bovine exosome, including 4 (N), 2 (O) and 2 (C) glycan binding sites; 298 proteins were identified in Caco‐2 cells, including 46 membrane proteins that included 29 (N), 33 (O) and 6 (C) glycan binding sites.ConclusionsGlycoproteins on the surface of bovine milk exosomes and intestinal cells facilitate the uptake of exosomes in human intestinal cells. The β‐galactoside, Core 1 & Core 3‐O‐linked disaccharide and N‐acetylglucosamine modifications appear to be of greater importance than other glycan features present on complex glycoproteins.Future DirectionsThe transport of bovine exosomes will be studied in conditional glycan knockout mice.Support or Funding InformationNIFA 2015‐67017‐23181, NIFA 2016‐67001‐25301/NIH DK107264, NIH 1P20GM104320, the Gerber Foundation, and USDA Hatch Act and W3002.