Abstract
UDP-glucose (UDP-Glc) is synthesized by UGP2-encoded UDP-Glc pyrophosphorylase (UGP) and is required for glycoconjugate biosynthesis and galactose metabolism because it is a uridyl donor for galactose-1-P (Gal1P) uridyltransferase. Chinese hamster lung fibroblasts harboring a hypomrphic UGP(G116D) variant display reduced UDP-Glc levels and cannot grow if galactose is the sole carbon source. Here, these cells were cultivated with glucose in either the absence or presence of galactose in order to investigate glycoconjugate biosynthesis and galactose metabolism. The UGP-deficient cells display < 5% control levels of UDP-Glc/UDP-Gal and > 100-fold reduction of [6-3H]galactose incorporation into UDP-[6-3H]galactose, as well as multiple deficits in glycoconjugate biosynthesis. Cultivation of these cells in the presence of galactose leads to partial restoration of UDP-Glc levels, galactose metabolism and glycoconjugate biosynthesis. The Vmax for recombinant human UGP(G116D) with Glc1P is 2000-fold less than that of the wild-type protein, and UGP(G116D) displayed a mildly elevated Km for Glc1P, but no activity of the mutant enzyme towards Gal1P was detectable. To conclude, although the mechanism behind UDP-Glc/Gal production in the UGP-deficient cells remains to be determined, the capacity of this cell line to change its glycosylation status as a function of extracellular galactose makes it a useful, reversible model with which to study different aspects of galactose metabolism and glycoconjugate biosynthesis.
Highlights
UDP-Glc is required for the biosynthesis of several types of glycoconjugate including glycogen, glycoproteins, glycosaminoglycans and glycosphingolipids (GSL) as well as the polysaccharide hyaluronan (Figure 1)
We could not detect dolichol-linked oligosaccharide (DLO) glucosylation in these cells (Durrant, C. and Moore, S., unpublished observations), and while attempting to boost UDP-glucose levels by supplementing the culture medium with mixtures of uridine and various monosaccharides, it was noted that supplementation of the culture medium with galactose alone was sufficient to allow complete DLO glucosylation (Durrant, C. and Moore, S., unpublished observations)
As we show that UDP-Glc/UDP-Gal levels in UDP-Glc pyrophosphorylase (UGP)− cells depend upon the ratio of glucose and galactose concentrations in the culture medium, the basal levels of these nucleotide sugars may be affected by the amounts of endogenous sources of galactose in the fetal calf serum used [10], especially after cells have depleted the glucose of the growth medium
Summary
UDP-Glc is required for the biosynthesis of several types of glycoconjugate including glycogen, glycoproteins, glycosaminoglycans and glycosphingolipids (GSL) as well as the polysaccharide hyaluronan (Figure 1). UDP-Glc is generated from glucose-1-phosphate (Glc1P) and UTP by UDP-Glc pyrophosphorylase (UGP). UGP is a critical regulator of cellular functions requiring either UDP-Glc or UDP-Gal (Figure 1). The mutant protein is called CgUGP(G116D) as the previous numbering did not take into account the initiating methionine residue. These cells have been reported to display 4% residual UGP activity, a ~70% reduction in UDP-Glc, and are unable to survive when galactose is presented as the sole energy source [5]. The mutant fibroblasts display altered epithelial-like morphology [7] and present with elevated glucose-regulated protein (GRP) expression [4]
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