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Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer.

ABSTRACT: Transepithelial transport of proteins is an important step in the immune response to food allergens. Mammalian meat allergy is characterized by an IgE response against the carbohydrate moiety galactosyl-?-1,3-galactose (?-Gal) present on mammalian glycoproteins and glycolipids, which causes severe allergic reactions several hours after red meat consumption. The delayed reaction may be related to the processing of ?-Gal carrying proteins in the gastrointestinal tract. The aim of this study was to investigate how protein glycosylation by ?-Gal affects the susceptibility to gastric digestion and transport through the Caco-2 cell monolayer. We found that ?-Gal glycosylation altered protein susceptibility to gastric digestion, where large protein fragments bearing the ?-Gal epitope remained for up to 2 h of digestion. Furthermore, ?-Gal glycosylation of the protein hampered transcytosis of the protein through the Caco-2 monolayer. ?-Gal epitope on the intact protein could be detected in the endosomal fraction obtained by differential centrifugation of Caco-2 cell lysates. Furthermore, the level of galectin-3 in Caco-2 cells was not affected by the presence of ?-Gal glycosylated BSA (bovine serum albumin) (BSA-?-Gal). Taken together, our data add new knowledge and shed light on the digestion and transport of ?-Gal glycosylated proteins.

SUBMITTER: Krstic Ristivojevic M

PROVIDER: S-EPMC7461108 | BioStudies | 2020-01-01

REPOSITORIES: biostudies

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Project description:A simple and efficient synthetic glycosylation method suitable for use in solid-phase enzymic oligosaccharide synthesis and site-specific glycosylation of recombinant proteins to produce defined glycoforms is described. This strategy utilizes N-(beta-saccharide) haloacetamides for attaching oligosaccharides specifically to cysteine residues of proteins in solution to form neoglycoproteins. The alkylation reaction was tested using N-(beta-chitotriose) bromoacetamide and an unprotected synthetic hexapeptide containing a single cysteine residue. The glycosylated product was confirmed by amino acid and hexosamine analyses as well as laser desorption mass spectrometry. Similarly N-(beta-chitotriose) iodoacetamide was covalently linked to non-reduced BSA to produce a defined glycoform of this protein. The specific attachment of chitotriose at the single cysteine residue in non-reduced serum albumin was suggested by Ellman's assay for free thiols. This was verified by amino acid sequencing of tryptic glycopeptide derived from this neoglycoprotein. Multiple sugar attachment was accomplished using fully reduced serum albumin as demonstrated by the formation of two neoglycoproteins using iodoacetamide derivatives of galactose beta 1-3-N-acetylgalactosamine (Gal beta 1-3GalNAc) and the major xylose/fucose-class plant-type oligosaccharide of horseradish peroxidase. These two neoglycoproteins with an average of 18-21 sugar residues attached were assayed positively for binding to peanut agglutinin and a sugar-specific anti-(horseradish peroxidase) monoclonal antibody YZ1/2.23 respectively. Sialylation of the neoglycoprotein containing Gal beta 1-3GalNAc was accomplished using alpha-2,3-sialyltransferase and radiolabelled CMP-N-acetylneuraminic acid. Significantly, glycan attachment using this conjugation method is reversible as demonstrated by the release of oligosaccharides from these two neoglycoproteins using hydrazinolysis. Therefore this method could provide invaluable reagents for many glycobiological studies.

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Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer.

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