UDP-Gal

Galactose Hexose Gal UDP-Gal Often found subterminal to NeuAc in N-linked glycoproteins. Also found in core trisaccharide of proteoglycans. 

Most nucleotide sugars are formed in the cytosol, generally from reactions involving the corresponding nucleoside triphosphate. GMP-sialic acids are formed in the nucleus. Formation of uridine diphosphate galactose (UDP-Gal) requires the following two reactions in mammahan tissues ... 

Membranes (50 pi in a total assay volume of 100 pi) were incubated with UDP-Gal (0.1 mM) and MgSO (10 mM) in 25 mM Tris-HCl buffer pH 7.5, for 10 or 60 min. Reactions were stopped by heating at 100°C for 3 min. Lupin galactan (0.1 mg) was added as a 0.1% solution, methanol was added to give a final concentration of 70% by volume, and the tubes were capped, heated at 70°C for 5 min and centrifuged (13000g 5 min). Supernatants were discarded or retained for analysis. Pellets were washed twice more with 70% methanol at 70 C and the supernatants were discarded. The final pellets were either dissolved in preparation for scintillation counting, or were suspended in water and freeze dried in preparation for analysis. 

Our standard incorporation assays contained resuspended particulate enzyme, labelled UDP-Gal (0.1 mM) and (10 mM) in resuspension buffer (Tris, pH 7.5). After incubation, reaction mixtures were heated briefly to 100°C and soluble lupin galactan was added, to ensure the precipitation of small amounts of galactan formed in the en me reaction and dissolved during the heating step. Precipitation of macromolecular products was achieved by adding methanol to a final concentration of 70%. The pellet was freed of soluble labelled products, including residual UDP-Gal, by repeated extraction with hot 70% methanol and was then analysed for labelled (l- )-P-D-galactan. The supernatant was analysed for soluble labelled products. 

Figure 2. Progress curve for the incorporation of label from UDP-Gal.

Figure 4. Digital autoradiogram of supernatants from time-points of Fig. 2. Lanes 1 - 10 = incubation times 0, 5, 15, 20, 30, 45, 60, 90, 120 min. Lane 11 = labelled UDP-Gal as reference.

Fig.8 Structure of UDP-Gal with labeling of the residues (G galactose, R ribose, U uracil) and torsion angles. Reprinted with permission from [69] (supporting information) 2004, American Chemical Society...

Table 3 Comparison of experimental and calculated percentage fractional STD intensities for UDP-Gal/GalTl complex...

Table 4 Comparison of torsion angles for different strnctnres of UDP-Gal/p4 GalTl complex...

In conclusion, using high-quality STD-NMR data on UDP-Gal weakly binding to 34 GalTl, we demonstrated that it is possible to refine the crystal structure (or any computer-docked structure in the proper orientation that serves as the starting structure) to obtain a global-minimum conformation for the bound ligand in solution. 

Acknowledgements This work was supported in part by the NCI grant CA-13148. The authors wish to thank Prof. B. Mario Pinto for supplying the originals of some figmes used in this review. The work on sialoadhesin/sialyllactose and UDP-Gal/galctosyltransferase complexes was from a fruitful collaboration with Prof. Thomas Peters at the Medical University of Liibeck. 

Scheme 5.7 Combination of three bacterial strains for the production of globotriose (13) with regeneration of UDP-Gal [40]. Combination of C. ammoniagenes DN510 (A), E. coli NM 522 pNT25/pNT32 (B) and E. coli NM522 pGT5 (C).

The first prominent example of large-scale oligosaccharide production by bacterial coupling was described by Koizumi et al. (Scheme 5.7) [40]. Three engineered bacterial strains were combined for the production of globotriose (13) including regeneration of UDP-Gal. 

For the preparation of p-o-galactopyranosyl (1 4)-2-acetamido-2-deoxy-p-D-gluco-pyranosyl-(l —>0)-elymoclavine (14) it was decided to extend previously prepared 2-acetamido-2-deoxy-p-D-glucopyranosyl-(1 0)-elymoclavine (15) using bovine p-l,4-galactosyltransferase. Uridine5 -diphosphogalactose (UDP-Gal) served as a substrate [48] (Fig. 11). 

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