A Transfected Sialyltransferase That Is Elevated in Breast Cancer and Localizes to the medial/trans-Golgi Apparatus Inhibits the Development of core-2–based O-Glycans

Whitehouse, Caroline; Burchell, Joy; Gschmeissner, Stephen; Brockhausen, Inka; Lloyd, Kenneth O.; Taylor-Papadimitriou, Joyce

Changes in the carbohydrate side chains of MUC1 expressed in the transfected MTSV1-7 and T47D cell lines were analyzed directly by high performance anion exchange chromatography (HPAEC) as previously described (Lloyd et al., 1996) . Briefly, cells were metabolically labeled with 100 Ci/ml [ 3 H]glucosamine-hydrochloride (Amersham Intl.) and MUC1 immunoprecipitated with CT1, an antibody to the cytoplasmic tail of MUC1 (Pemberton et al., 1992) . The carbohydrate side chains were released by alkaline borohydride treatment. Samples containing 10,000 cpm were then separated on a Carbo Pak PA1 column (Dionex Corp., Sunnyvale, CA) using a gradient of 0.2 M NaOH to 0.2 M NaOH-0.25 M sodium acetate at 1.0 ml/min over 30 min (Lloyd and Savage, 1991) . Collected radioactive fractions were neutralized with 1 M HCl before counting. For hexosamine analysis, the immunoprecipitate was eluted in 2% SDS and hydrolyzed in 2 N trifluoroacetic acid at 100 Њ C for 3 h, and 5,000 cpm samples were analyzed by HPAEC on a CarboPak PA1 column by isocratic elution with 0.01 M NaOH at 1.0 ml/min.