Synthesis and evaluation of mimetics of UDP and UDP-alpha-D-galactose, dTDP and dTDP-alpha-D-glucose with monosaccharides replacing the key pyrophosphate unit.

A series of 5'-O-glycosyl-uridine and thymidine derivatives have been prepared as potential mimics of sugar nucleotides and nucleotide-diphosphates. These compounds proved not to be inhibitors of bovine beta-1,4-galactosyltransferase although some showed moderate inhibition of Salmonella dTDP-alpha-D-glucose 4,6-dehydratase (RmlB).

T lymphocyte activation results in an increased expression of beta-1,4-galactosyltransferase: phorbol ester induces a similar enhancement in the absence of mitosis.

We previously showed that in vitro activated human T lymphocytes expressed increased amounts of beta-1,6-branched N-linked oligosaccharides (Lemaire S etal. (1994) J Biol Chem269: 8069-74), which have been proposed to participate in the regulation of the immune process. In the present paper, we compared the activity and expression of beta-1,4-galactosyltransferase (GalT), one of the glycosyltransferases involved in the biosynthesis of these beta-1,6-branched N-linked oligosaccharides, before and after in vitro activation of T lymphocytes after a 40h treatment with a mixture of phorbol 12-myristate 13-acetate and Phaseolus vulgaris lectin. After treatment, the enzymatic activity of the GalT was significantly increased and immunoblot experiments performed with a monoclonal antibody to human GalT showed an increased intensity of the GalT band at 49 kDa, attributable to an enhancement of GalT mRNA level, as shown by Northern blots. However, treatment of the same T-lymphocytes by phorbol ester alone, which is unable to induce mitosis, resulted in a comparable increase of the expression of GalT. Moreover, these phorbol ester-treated T lymphocytes, analysed by flow cytometry exhibited a two-fold increase in the expression of GalT. Finally, confocal fluorescence microscopy performed on all T lymphocytes (treated or not) showed that the flow cytometric signal of GalT originates from intracellular, Golgi-associated antigen only since no surface GalT was detected.

Alpha-lactalbumin induces bovine milk beta 1,4-galactosyltransferase to utilize UDP-GalNAc.

We now report that alpha-lactalbumin (alpha-LA) has a novel effect on bovine milk UDP-Gal:GlcNAc-beta 1,4-galactosyltransferase (beta 1,4-GT) and induces the enzyme to efficiently utilize UDP-GalNAc as a donor. In the presence of alpha-LA the enzyme transfers GalNAc to free GlcNAc to produce GalNAc beta 1-4GlcNAc at a rate 55% of that compared to the rate when UDP-Gal is the donor in the absence of alpha-LA. The stimulation by alpha-LA is dependent on the concentrations of alpha-LA, acceptor, and sugar nucleotide. Interestingly, beta 1,4-GT is unable to transfer Gal-NAc to Glc with or without alpha-LA. alpha-LA also stimulates the transfer of GalNAc from UDP-GalNAc to various chitin oligomers, although the degree of stimulation decreases as the acceptor size increases. Thus, bovine milk beta 1,4-GT has an inherent ability to utilize two different sugar nucleotides and the sugar nucleotide preference is regulatable by alpha-LA.

Double immunofluorescent staining of alpha 2,6 sialyltransferase and beta 1,4 galactosyltransferase in monensin-treated cells: evidence for different Golgi compartments?

Beta 1,4 galactosyl- and alpha 2,6 sialyltransferase (gal-T EC 2.4.1.22 and sialyl-T EC 2.4.99.1) sequentially elongate and terminate complex N-glycan chains of glycoproteins. Both enzymes reside in trans Golgi cisternae; their ultrastructural relationship, however, is unknown. To delineate their respective Golgi compartment(s) we conducted a double label immunofluorescent study by conventional and confocal laser scanning microscopy in HepG2, HeLa, and other cells in presence of Golgi-disturbing agents. Polyclonal, peptide-specific antibodies to human sialyl-T expressed as a beta-galactosidase-sialyl-T fusion protein in E. coli were developed and applied together with mABs to human milk gal-T. In untreated HepG2 and HeLa cells Golgi morphology identified by immunofluorescent labeling of sialyl-T and gal-T, respectively, was nearly identical. Treatment of cells with brefeldin A (BFA) led to rapid and coordinated disappearance of immunostaining of both enzymes; after BFA washout, vesicular structures reappeared which first stained for gal-T followed by sialyl-T; in the reassembled Golgi apparatus sialyl-T and gal-T were co-localized again. In contrast, monensin treatment produced a reversible swelling and scattering of gal-T positive Golgi elements while sialyl-T positive structures showed little change. Treatment with nocodazole led to dispersal of Golgi elements in which gal-T and sialyl-T remained co-localized. Treatment with chloroquine affected Golgi structures less than monensin and led to condensation of gal-T positive and to slight enlargement of sialyl-T positive structures. Sequential recovery from BFA of gal-T and sialyl-T and their segregation by monensin suggest that these enzymes are targeted to different Golgi subcompartments.