Characterization of mucins and proteoglycans synthesized by a mucin-secreting HT-29 cell subpopulation.
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.
Huet
G
G
INSERM U377, Lille, France.
Kim
I
I
de Bolos
C
C
Lo-Guidice
J M
JM
Moreau
O
O
Hemon
B
B
Richet
C
C
Delannoy
P
P
Real
F X
FX
Degand
P
P
D000116:Acetylgalactosamine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D001593:Benzyl Compounds / Q000378:metabolism / Q000494:pharmacology; D020126:Brefeldin A; D002240:Carbohydrate Sequence; D003110:Colonic Neoplasms / Q000378:metabolism; D003517:Cyclopentanes / Q000494:pharmacology; D004187:Disaccharides / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009077:Mucins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D011509:Proteoglycans / Q000096:biosynthesis* / Q000737:chemistry; D012794:Sialic Acids / Q000378:metabolism; D012799:Sialyltransferases / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D001593:Benzyl Compounds; D003517:Cyclopentanes; D004187:Disaccharides; D009077:Mucins; D011509:Proteoglycans; D012794:Sialic Acids; C090985:benzyl 2-acetamido-2-deoxy-3-O-galactopyranosylgalactopyranoside; D020126:Brefeldin A; C080739:benzyl-alpha-N-acetylgalactosamine; D012799:Sialyltransferases; D000116:Acetylgalactosamine; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase; C000722087:ST3GAL1 protein, human