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Characterization of mucins and proteoglycans synthesized by a mucin-secreting HT-29 cell subpopulation
1INSERM U377, Lille, France.
Abstract:
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.
Insights
Methotrexate-adapted HT-29 MTX cells are a valuable model for studying mucin synthesis. These cells produce high amounts of mucin, and their sialylation can be modulated by specific inhibitors.
Area of Science:
- Biochemistry
- Cell Biology
- Glycobiology
Background:
- HT-29 cells adapted to methotrexate (HT-29 MTX) form a homogeneous population with high mucin production.
- Understanding mucin synthesis and glycosylation is crucial in various biological processes.
Purpose of the Study:
- To characterize mucins and proteoglycans from HT-29 MTX cells.
- To investigate the effects of brefeldin A and GalNAc-alpha-O-benzyl on mucin synthesis and properties.
- To establish HT-29 MTX cells as a model for analyzing mucin synthesis and sialylation.
Main Methods:
- Isolation and ultracentrifugation of intracellular mucins and proteoglycans.
- Anion-exchange high-performance liquid chromatography for separation.
- Biochemical characterization of isolated mucin fractions.
- Treatment with brefeldin A and GalNAc-alpha-O-benzyl to study synthesis effects.
Main Results:
- HT-29 MTX cells yielded a major mucin fraction rich in hydroxy amino acids, sialic acid, and low in sulfate.
- Undifferentiated HT-29 cells produced less sialylated and more sulfated mucins.
- GalNAc-alpha-O-benzyl treatment significantly reduced sialic acid content and increased T antigen, acting as an inhibitor of O-glycan alpha-2,3-sialyltransferase.
Conclusions:
- HT-29 MTX cells provide a robust model for studying mucin biosynthesis.
- Specific inhibitors like GalNAc-alpha-O-benzyl can modulate mucin sialylation.
- The study elucidates key aspects of mucin synthesis and glycosylation pathways.