Novel O-linked glycans containing 6'-sulfo-Gal/GalNAc of MUC1 secreted from human breast cancer YMB-S cells: possible carbohydrate epitopes of KL-6(MUC1) monoclonal antibody

Human serum Krebs von den Lugen-6 (KL-6) antigen is a MUC1 glycoprotein (KL-6/MUC1) recognized by anti-KL-6 monoclonal antibody (KL-6/mAb) and has been utilized as a diagnostic marker for interstitial pneumonia. KL-6/mAb is thought to recognize the specific glycopeptides sequence of MUC1, but the precise glycan structure of the epitope is unclear. In this study, we determined the carbohydrate structures of KL-6/MUC1 to search the carbohydrate epitopes for KL-6/mAb. KL-6/MUC1 was purified from the culture medium of human breast cancer YMB-S cells by KL-6/mAb-affinity chromatography; the O-linked glycan structures were determined in combination with paper electrophoresis, several lectin column chromatographies, sialidase digestion and methanolysis. KL-6/MUC1 contained core 1 and extended core 1 glycans modified with one or two sialic acid/sulfate residues. Based on these structures, several synthetic glycans binding to anti-KL-6/mAb were compared with one another by surface plasmon resonance. Sequentially, related radiolabeled oligosaccharides were enzymatically synthesized and analyzed for binding to a KL-6/mAb-conjugated affinity column. 3'-sialylated, 6'-sulfated LNnT Neu5Acα2-3(SO(3)(-)-6)Galβ1-4GlcNAcβ1-3Galβ1-4Glc], 3'-sialylated, 6-sulfated core 1 Neu5Acα2-3Galβ1-3(SO(3)(-)-6)GalNAc] and disulfated core 1 SO(3)(-)-3Galβ1-3(SO(3)(-)-6)GalNAc exhibited substantial affinity for KL-6/mAb, and 3'-sulfated core 1 derivatives SO(3)(-)-3Galβ1-3(±Neu5Acα2-6)GalNAc] and 3'-sialylated core 1 weakly interacted with KL-6/mAb. These results indicated that the possible carbohydrate epitopes of KL-6/mAb involve not only 3'-sialylated core 1 but also novel core 1 and extended core 1 with sulfate and sialic acid residues. Epitope expressing changes with suppression or over-expression of the Gal6ST (Gal 6-O-sulfotransferase) gene, suggesting that Gal6ST is involved in the biosynthesis of the unique epitopes of KL-6/mAb.