G. Huet et al., CHARACTERIZATION OF MUCINS AND PROTEOGLYCANS SYNTHESIZED BY A MUCIN-SECRETING HT-29 CELL SUBPOPULATION, Journal of Cell Science, 108, 1995, pp. 1275-1285
HT-29 cells selected by adaptation to 10(-5) M methotrexate (HT-29 MTX
) are a homogeneous cell population producing high amounts of mucin, I
ntracellular mucins and proteoglycans were isolated from these cells b
y ultracentrifugation of cell lysates on a cesium bromide gradient and
further separated by anion-exchange high perfomance liquid chromatogr
aphy. The major mucin fraction isolated was characterized by a high hy
droxy 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 is
olated which appeared less sialylated and more sulfated, The major pro
teoglycan species identified in HT-29 MTX cells showed less acidic beh
avior than the proteoglycan isolated from HT-29 cells. The effect of b
refeldin A and the sugar analog GalNAc-alpha-O-benzyl on the synthesis
and biochemical properties of mucins synthesized by HT-29 MTX cells w
as 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 inhi
bitor of the O-glycan alpha-2,3-sialyltransferase. These results illus
trate the suitability of HT-29 MTX cells as a model to analyse mucin s
ynthesis and sialylation.