Inhibition of protein N-glycosylation by 2-deoxy-2-fluoro-d-galactose

Abstract

The effects of 2-deoxy-2-fluoro-D-galactose (dGalF) on N- and O-glycosylation of proteins was studied in rat hepatocyte primary cultures and in human monocytes. In hepatocytes, dGalF at concentrations of 1 mM or higher completely inhibited N-glycosylation of alpha 1-antitrypsin and alpha 1-acid glycoprotein, whereas 4 mM-2-deoxy-D-galactose (dGal) only slightly impaired N-glycosylation. In monocytes, 1 mM- or 4 mM-dGalF blocked N-glycosylation of alpha 1-antitrypsin and of interleukin-6, while O-glycosylation of interleukin-6 remained unaffected. In monocytes, dGal had no effect on protein N-glycosylation. Addition of uridine effectively prevented the UTP deficiency induced by dGalF, but had no effect on the inhibition of protein N-glycosylation by dGalF. Using 19F-n.m.r. spectroscopy, 2-deoxy-2-fluoro-D-galactose 1-phosphate (dGalF-1-P), UDP-dGalF and UDP-dGlcF could be identified as the major metabolites of dGalF in hepatocytes as well as in monocytes. In conclusion, compared with dGal, dGalF is a more efficient inhibitor of protein N-glycosylation. The effect is not caused by the depletion of UTP induced by dGalF, but rather by metabolites of dGalF. dGalF is metabolized not only in hepatocytes but also in peripheral blood monocytes, which can be used for ex vivo studies of disturbances in D-galactose metabolism.

Fluorinated Galactoses Inhibit Galactose-1-Phosphate Uridyltransferase and Metabolically Induce Galactosemia-like Phenotypes in HEK-293 Cells

“…We were not able to demonstrate effects on N-glycosylation of doxycyclin-inducible netrin-1 expressed in HEK293 cells in the presence of fluorinated galactose. These results obtained by mass spectrometric N-glycan profiling are in striking contrast to previous reports of two groups that were claiming a more or less complete abrogation of N-glycosylation in the presence of fluorinated galactose above 1 mM [19,20]. However, it has to be pointed out that these effects may be cell-type restricted and cannot be expected to play a major role in HEK293 cells.…”

“…In reference to previous work demonstrating a dys-glycosylation induced by fluorinated galactose [19,20], we analyzed a recombinant secretory glycoprotein probe for effects on N-glycosylation in transfected HEK293 by mass spectrometry. Previous work had demonstrated a more or less complete inhibition of N-glycosylation (between 1 to 5 mM F-Gal), when cells had been grown in the presence of fluorinated galactose.…”

“…In this way, fluorinated galactitol can be formed by the aldose reductase with concomitant depletion of the NADPH pool (Figure 9). Obviously, in HEK293 cells, the latter pathways play more prominent roles than the conversion of F-Gal to the UDP-activated intermediates that may ultimately lead to the formation of Dol-PP-derivatives of F-Gal and to a block of N-glycosylation [20]. .…”