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Journal of Cell Science, Vol 108, Issue 3 1275-1285, Copyright © 1995 by Company of Biologists
JOURNAL ARTICLES |
G Huet, I Kim, C de Bolos, JM Lo-Guidice, O Moreau, B Hemon, C Richet, P Delannoy, FX Real and P Degand
INSERM U377, Lille, France.
HT-29 cells selected by adaptation to 10(-5) M methotrexate (HT-29 MTX) are a homogeneous cell population producing high amounts of mucin. Intracellular mucins and proteoglycans were isolated from these cells by ultracentrifugation of cell lysates on a cesium bromide gradient and further separated by anion-exchange high performance liquid chromatography. The major mucin fraction isolated was characterized by a high hydroxy amino acid content (40%), a Thr/Ser ratio of 1.52, a high sialic acid content, and a low sulfate content. When the same procedure was applied to undifferentiated HT-29 cells, a minor mucin fraction was isolated which appeared less sialylated and more sulfated. The major proteoglycan species identified in HT-29 MTX cells showed less acidic behavior than the proteoglycan isolated from HT-29 cells. The effect of brefeldin A and the sugar analog GalNAc-alpha-O-benzyl on the synthesis and biochemical properties of mucins synthesized by HT-29 MTX cells was examined. Brefeldin A induced the synthesis of more-sulfated mucins. GalNAc-alpha-O-benzyl treatment resulted in mucins with an increased content of T antigen and a 13-fold lower sialic acid content. We show that GalNAc-alpha-O-benzyl was metabolized by the cells to Gal beta 1-3GalNAc-alpha-O-benzyl, which, in turn, was a potent competitive inhibitor of the O-glycan alpha-2,3-sialyltransferase. These results illustrate the suitability of HT-29 MTX cells as a model to analyse mucin synthesis and sialylation.
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