Characterization of Carbohydrate Structures of Bovine MUC15 and Distribution of the Mucin in Bovine Milk

Proteolysis of MUC15 by the O-sialoglycoprotein endo-peptidase of Mannheimia haemolytica. Western blot probed with polyclonal antibodies against bovine MUC15. Lane 1, bovine MUC15 control; lane 2, MUC15 treated with O-sialoglycoprotein endopeptidase for 15min; lane 3, MUC15 treated with O-sialoglycoprotein endopeptidase for 2.5h; lane 4, MUC15 treated with O-sialoglycoprotein endopeptidase for 21h. Positions of molecular weight markers are indicated to the left.

Enzymatic deglycosylation of bovine MUC15. Analysis was performed on 18% Tris-glycine gels and stained with periodic acid Schiff's reagent. Lane 1, bovine MUC15; lane 2, neuraminidase-treated MUC15; lane 3, O-glycosidase-treated MUC15; lane 4, neuraminidase and O-glycosidase-treated MUC15. Positions of molecular weight markers are indicated to the left.

Enzymatic deglycosylation of bovine MUC15. Analysis was performed on 18% Tris-glycine gels. A) Gel stained with periodic acid Schiff's reagent and Coomassie Brilliant Blue. B) Western blot probed with polyclonal antibodies against bovine MUC15. Lane 1, bovine MUC15; lane 2, peptide:N-glycosidase F (PNGase F)-treated MUC15; lane 3, PNGase F- and neuraminidase-treated MUC15; lane 4, PNGase F-, neuraminidase-, and O-glycosidase-treated MUC15; lane 5, PNGase F-, neuraminidase-, O-glycosidase-, and β(1–4)-galactosidase-treated MUC15; lane 6, PNGase F-, neuraminidase-, O-glycosidase-, and β-N-acetylglucosaminidase-treated MUC15; lane 7, bovine MUC15 treated with all 5 enzymes. Positions of molecular weight markers are indicated to the left.

Immunoblot analysis of MUC15 from bovine milk with lectins of the Digoxigenin (DIG) Glycan Differentiation kit (Roche Diagnostics, Mannheim, Germany). Protein samples were separated by SDS-PAGE on 18% Tris-glycine polyacrylamide gels and blotted onto Hybond-P polyvinylidene difluoride membrane. Blots were sequentially incubated with DIG-labeled lectins, alkaline phosphatase-conjugated anti-DIG antibody, and nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate as described in the materials and methods section. A) Galanthus nivalis agglutinin (GNA); B) peanut agglutinin (PNA); C) Datura stramonium agglutinin (DSA); D) Maackia amurensis agglutinin (MAA); E) Sambucus nigra agglutinin (SNA). Two micrograms of MUC15 was used in each protein sample except in B, lanes 3 and 4, where 0.2μg was used. Lane 1, MUC15; lane 2, PNGase F-treated MUC15; lane 3, neuraminidase-treated MUC15; lane 4, neuraminidase- and PNGase F-treated MUC15. Positions of molecular weight markers are indicated to the left of each gel.