Reactivity of mucin-specific lectin from Sambucus sieboldiana with simple sugars, normal mucins and tumor-associated mucins. Comparison with other lectins.
Mucin-specific lectin from Sambucus sieboldiana (SSA-M) reacts in Western blotting and ELISA with mucins from porcine stomach, bovine and ovine submaxillary glands, the human milk fat globule membrane, in vitro human ovarian, breast and colonic tumor cell lines, and mucins produced in vivo in the ascites of patients with endometrial and ovarian tumors, but not with fetal bovine fetuin or human transferrin. Sialidase treatment of these mucins led to an increase in the binding of SSA-M, suggesting that sialic acid is not part of the binding site for this lectin. Furthermore, sialic acid did not inhibit lectin binding. Treatment of asialomucin with O-glycanase decreased the binding of SSA-M, confirming the reactivity of the lectin with an O-linked carbohydrate. Treatment of mucins with trifluoromethanesulfonic acid, which removes all but core carbohydrate, led to an increase in the binding of SSA-M, suggesting that the lectin reacts with O-linked core glycans. Indeed, the increased reactivity after sialidase treatment of ovine submaxillary mucin suggests the lectin reacts with peptide-linked N-acetylgalactosamine (GalNAc), since more than 98% of the glycan chains attached to this mucin are sialylated GalNAc. The binding of SSA-M to sialidase-treated porcine mucin was inhibited strongly by GalNAc and disaccharides containing galactose (lactose, melibiose, and N-acetyllactosamine) but not by free galactose (Gal), suggesting that the glycan for optimum binding is Gal beta(1-3)GalNAc. This pattern of inhibition was different to other core glycan-reactive lectins tested, indicating that SSA-M is distinct, and should be of use in the isolation and characterisation of mucins and O-linked glycans.
Devine
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Medical Innovations Limited, Labrador, Australia.
Harada
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D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D015153:Blotting, Western; D050260:Carbohydrate Metabolism; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D008698:Mesylates / Q000378:metabolism; D009077:Mucins / Q000378:metabolism*; D009439:Neuraminidase / Q000378:metabolism; D037121:Plant Lectins; D010944:Plants / Q000737:chemistry*; D054788:Ribosome Inactivating Proteins; D013552:Swine; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D006023:Glycoproteins; D037102:Lectins; D008698:Mesylates; D009077:Mucins; D037121:Plant Lectins; C050600:Sambucus nigra lectins; D006026:Glycoside Hydrolases; C020668:glycanase; D009439:Neuraminidase; D054788:Ribosome Inactivating Proteins; C012077:trifluoromethanesulfonic acid