Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.
The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.
Higuchi
T
T
Department of Biochemistry and Molecular Biology, Milton S. Hershey Medical Center, PA 17033, USA.
Xin
P
P
Buckley
M S
MS
Erickson
D R
DR
Bhavanandan
V P
VP
D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000276:immunology / Q000302:isolation & purification*; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004847:Epithelial Cells / Q000737:chemistry; D007150:Immunohistochemistry; D037102:Lectins; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000276:immunology / Q000302:isolation & purification*; D009092:Mucous Membrane / Q000737:chemistry; D037121:Plant Lectins; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013402:Sugar Alcohols / Q000737:chemistry; D001743:Urinary Bladder / Q000737:chemistry*
D015295:Antigens, Tumor-Associated, Carbohydrate; D037102:Lectins; D018396:Mucin-1; D037121:Plant Lectins; D013402:Sugar Alcohols; C043696:jacalin