Defects in Galactose Metabolism and Glycoconjugate Biosynthesis in a UDP-Glucose Pyrophosphorylase-Deficient Cell Line Are Reversed by Adding Galactose to the Growth Medium.

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.

Perturbation of free oligosaccharide trafficking in endoplasmic reticulum glucosidase I-deficient and castanospermine-treated cells.

Free oligosaccharides (FOS) are generated both in the endoplasmic reticulum (ER) and in the cytosol during glycoprotein biosynthesis. ER lumenal FOS possessing the di-N-acetylchitobiose moiety at their reducing termini (FOSGN2) are exported into the cytosol where they, along with their cytosolically generated counterparts possessing a single N-acetylglucosamine residue at their reducing termini (FOSGN1), are trimmed in order to be imported into lysosomes for final degradation. Both the ER and lysosomal FOS transport processes are unable to translocate triglucosylated FOS across membranes. In the present study, we have examined FOS trafficking in HepG2 cells treated with the glucosidase inhibitor castanospermine. We have shown that triglucosylated FOSGN2 generated in the ER are transported to the Golgi apparatus where they are deglucosylated by endomannosidase and acquire complex, sialic acid-containing structures before being secreted into the extracellular space by a Brefeldin A-sensitive pathway. FOSGN2 are also secreted from glucosidase I-deficient Lec23 cells and from the castanospermine-treated parental Chinese-hamster ovary cell line. Despite the secretion of FOSGN2 from Lec23 cells, we noted a transient intracellular accumulation (60 nmol/g cells) of triglucosylated FOSGN1 in these cells. Finally, in glucosidase I-compromised cells, FOS trafficking was severely perturbed leading to both the secretion of FOSGN2 into the extracellular space and a growth-dependent pile up of triglucosylated FOSGN1 in the cytosol. The possibility that these abnormalities contributed to the severe and rapidly progressive pathology in a patient with congenital disorders of glycosylation type IIb (glucosidase I deficiency) is discussed.