Characterization of two novel sialyl N-acetyllactosaminyl nucleotides separated from ovine colostrum.

The milk/colostrum of some mammalian species is known to contain sugar nucleotides including uridine diphosphate (UDP) oligosaccharides in addition to lactose and milk oligosaccharides, but the detailed structures of these UDP oligosaccharides have not so far been clarified. In this study we isolated two UDP-sialyl N-acetyllactosamines from ovine colostrum and characterized them using (1)H-NMR and MALDI-TOFMS spectroscopies. Their structures were found to be Neu5Gc(α2-3)Gal(ß1-4)GlcNAcα1-UDP and Neu5Gc(α2-6)Gal(ß1-4)GlcNAcα1-UDP.

Hexosamines are unlikely to function as a nutrient-sensor in 3T3-L1 adipocytes: a comparison of UDP-hexosamine levels after increased glucose flux and glucosamine treatment.

Whether the hexosamine biosynthesis pathway acts as a nutrient-sensing pathway is still unclear. Glucose is directed into this pathway by GFAT. Because the activity of GFAT is tightly regulated, we examined whether UDP-hexosamine levels can increase significantly and dose-dependently in response to elevated glucose concentrations. In glucosamine-treated 3T3-L1 adipocytes, inhibition of insulin-stimulated glucose uptake was highly correlated with UDP-hexosamine levels (r = -0.992; p < 0.0001 for UDP-GlcNAc and r = -0.996; p < 0.0001 for UDP-GalNAc). Incubation of 3T3-L1 adipocytes with 0.1 microM insulin for 24 h in medium containing 1 and 5 mM glucose increased the rate of glucose uptake by 365% and 175% compared to untreated cells, respectively. This increase was not observed when the cells were incubated for 24 h with insulin in medium containing 10 or 25 mM glucose. However, treatment of cells with insulin and 1, 5, 10, or 25 mM glucose resulted in similar increases in levels of UDP-GlcNAc and UDP-GalNAc that always amounted to approx 30-40% above baseline values. This led us to conclude that despite exposure of adipocytes to conditions of extreme and prolonged glucose disposal, the increases in cellular UDP-hexosamines were minimal and not dependent on the extracellular glucose concentration. Taken together, our results are in line with the hypothesis that in glucosamine-treated adipocytes UDP-hexosamines influence insulin-stimulated glucose uptake. However, our observations in glucose-treated adipocytes argue against the possibility that UDP-hexosamines function as a nutrient-sensor, and question the role of the hexosamine biosynthesis pathway in the pathogenesis of insulin resistance.

Effects of ammonia and glucosamine on the heterogeneity of erythropoietin glycoforms.

Recombinant human erythropoietin (EPO) is a glycoprotein produced as a therapeutic agent from mammalian cell cultures for the treatment of anemia associated with severe kidney damage. The EPO structure has a high glycan content which is essential for bioactivity but shows considerable molecular heterogeneity. The cell culture conditions that affect the heterogeneity of the glycoforms of EPO are not well understood. However, the accumulation of ammonia in culture is one factor that has been associated with an enhanced heterogeneity of glycoforms. In this report we investigate the metabolic perturbations associated with ammonia and glucosamine that may give rise to an altered pattern of EPO glycosylation. Recombinant human erythropoietin was synthesized in serum-free cultures of transfected Chinese hamster ovary (CHO) cells. The molecular heterogeneity of erythropoietin was increased by supplementation of cultures with either ammonia or glucosamine. The enhanced molecular heterogeneity was shown to be due to variable glycosylation that resulted in EPO with an enhanced molecular weight and isoelectric point range. Enzymatic removal of the glycan moiety of EPO in all cases resulted in a single molecular form with a molecular weight of 18 000, which corresponded to non-glycosylated EPO. The variable glycosylation was consistent with reduced sialylation and antennarity of the carbohydrate structures present on the three N-linked sites of EPO. In the presence of ammonia (>30 mM) the proportion of tetrasialylated and tetraantennary glycan structures were reduced by 73% and 57%, respectively, as determined by HPLC analysis. Such changes were also observed, although to a lesser extent (41% and 37%), by an increase in the glucosamine concentration (>10 mM) in the medium. The enhanced heterogeneity of the glycan structures coincided with a significant increase in the intracellular UDP-N-acetylhexosamine (UDP-GNAc) pool. The measured UDP-GNAc level was up to 2 orders of magnitude higher in the presence of either glucosamine or ammonia. However, the changes in the glycosylation profiles induced by either glucosamine or ammonia were significantly different even at the same intracellular UDP-GNAc concentration. This suggests that the enhanced EPO heterogeneity could not be mediated solely by the increased UDP-GNAc level. Glucosamine (but not ammonia) was shown to cause significant inhibition of glucose transport into the cells, which could induce a different pattern of primary metabolism.

Vicia villosa B4 lectin inhibits nucleotide pyrophosphatase activity toward UDP-GalNAc specifically.

Plant seed lectins play a defense role against plant-eating animals. Here, GalNAc-specific Vicia villosa B4 lectin was found to inhibit hydrolysis of UDP-GalNAc by animal nucleotide pyrophosphatases, which are suggested to regulate local levels of nucleotide sugars in cells. Inhibition was marked at low concentrations of UDP-GalNAc, and was reversed largely by the addition of GalNAc to the reaction mixture. In contrast, lectin inhibited enzymatic hydrolysis of other nucleotide sugars, such as UDP-Gal and UDP-GlcNAc, only to a small extent, and GalNAc did not affect such an inhibition. The binding constant of the lectin for UDP-GalNAc was as high as 2.8 x 10(5) M-1 at 4 degrees C, whereas that for GalNAcalpha-1-phosphate was 1.3 x 10(5) M-1. These findings indicate that lectin inhibition of pyrophosphatase activity toward low concentrations of UDP-GalNAc arises mainly from competition between lectin and enzyme molecules for UDP-GalNAc. This type of inhibition was also observed to a lesser extent with GalNAc-specific Wistaria floribunda lectin, but not apparently with GalNAc-specific soybean or Dolichos biflorus lectin. Thus, V. villosa B4 lectin shows unique binding specificity for UDP-GalNAc and has the capacity to modulate UDP-GalNAc metabolism in animal cells.

Discrimination of fluorinated uridine metabolites in N-417 small cell lung cancer cell extracts via 19F- and 31P-NMR.

31P-NMR extract spectra of N-417 Small Cell Lung Cancer (SCLC) cells cultured with fluorouridine (FUrd) reveal new peaks with chemical shifts in the diphosphodiester and nucleoside triphosphate regions. These peaks were identified as FUTP, FUDP, FUDP-glucose, FUDP-glucuronate, FUDP-GlcNAc, and FUDP-GalNAc via enzymatic conversion and 19F- and 31P-NMR analysis. Distinct 19F chemical shifts were assigned for FUTP, FUDP, and the FUDP-sugars.

Human retinal pigment epithelial cells cultured in hyperglycemic media accumulate increased amounts of glycosaminoglycan precursors.

Confluent human retinal pigmented epithelial cells were cultured on microcarrier beads in the presence of 5.6 or 26 mmol/l glucose with or without the aldose-reductase inhibitor Sorbinil (200 microM) for 2 wk. At the end of the incubation period, perchloric acid extracts were prepared and analyzed by 31P nuclear magnetic resonance spectroscopy. As assessed by this method, the phosphorylated metabolites of cells incubated with 5.6 or 26 mmol/l glucose differed significantly in the concentrations of a number of uridine diphosphate (UDP)-conjugated monosaccharides, which were elevated two- to threefold in cells incubated in 26 mmol/l glucose over control samples. The affected metabolites were identified (through a series of spiking experiments) to be UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, and UDP-glucuronic acid. Coincubation of the cells with Sorbinil 200 microM in the presence of 26 mmol/l glucose had no effect on this accumulation. Under normal circumstances, these molecules selectively and sequentially are incorporated into the polysaccharide chains of glycosaminoglycans (GAGs), whose presence and distribution in the basement membranes is affected adversely by diabetes mellitus. These data suggest that the availability of the monosaccharide precursor is not the rate-limiting step for GAG synthesis in the presence of pathologic glucose concentrations. Thus, the lost GAG content in the basement membranes of diabetic patients may be caused by changes elsewhere in the biosynthesis and/or catabolism of the polysaccharide-linked protein molecules.

Phosphorus metabolite characterization of human prostatic adenocarcinoma in a nude mouse model by 31P magnetic resonance spectroscopy and high pressure liquid chromatography.

A series of experiments were conducted to identify and quantify the phosphorus metabolites of DU 145 xenografts (a human prostatic adenocarcinoma cell line grown in nude mice) using 31P MRS and HPLC. The 31P spectral characteristics of DU 145 xenografts were compared to perfused DU 145 cells and to in situ human prostatic adenocarcinomas. These studies demonstrated that both DU 145 xenografts and perfused DU 145 cells exhibited reduced levels of phosphocreatine relative to spectra of in situ human prostatic adenocarcinomas. Elevated levels of phosphomonesters (PMEs) were observed in 31P spectra of both DU 145 xenografts and in situ human prostatic adenocarcinomas. The major components of the PME resonance of DU 145 xenografts were identified as phosphocholine and phosphoethanolamine. High levels of diphosphodiesters (DPDEs) were consistently observed for both DU 145 xenografts and perfused DU 145 cells, but were absent in 31P spectra in in situ primary human adenocarcinomas. In agreement with spectroscopic results, high pressure liquid chromatographic analyses of human tissue removed at surgery contained insignificant amounts of DPDEs while DU 145 xenografts had high levels of DPDEs consisting mainly of uridine-5'-diphospho-N-acetylgalactosamine (22.4 nmol/mg protein) and uridine-5'-diphospho-N-acetylglucosamine (7.4 nmol/mg protein).

The influence of flanking sequences on O-glycosylation.

The influence of flanking sequences on O-glycosylation of serine and threonine residues was explored by comparison of known acceptor sites. Positions -6, -1 and +3 relative to the site were identified as particularly significant. To test the hypothesis that O-glycosylation could be affected by amino acid sequence, a series of test peptides was made containing substitutions at the sensitive positions. In vitro glycosylation of the peptides confirmed that the acceptor status of threonine was markedly influenced by the residues present at positions -6, -1 and +3. Circular dichroism indicated that peptides which had random structure were glycosylated, except when they contained a charged residue at position -1.