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Homologous glycosyltransferases GTA and GTB perform the final step in human ABO(H) blood group A and B antigen synthesis by transferring the sugar moiety from donor UDP-GalNAc/UDP-Gal to the terminal H antigen disaccharide acceptor. Like other GT-A fold family 6 glycosyltransferases, GTA and GTB undergo major conformational changes in two mobile regions, the C-terminal tail and internal loop, to achieve the closed, catalytic state. These changes are known to establish a salt bridge network among conserved active site residues Arg188, Asp211 and Asp302, which move to accommodate a series of discrete donor conformations while promoting loop ordering and formation of the closed enzyme state. However, the individual significance of these residues in linking these processes remains unclear. Here, we report the kinetics and high-resolution structures of GTA/GTB mutants of residues 188 and 302. The structural data support a conserved salt bridge network critical to mobile polypeptide loop organization and stabilization of the catalytically competent donor conformation. Consistent with the X-ray crystal structures, the kinetic data suggest that disruption of this salt bridge network has a destabilizing effect on the transition state, emphasizing the importance of Arg188 and Asp302 in the glycosyltransfer reaction mechanism. The salt bridge network observed in GTA/GTB structures during substrate binding appears to be conserved not only among other Carbohydrate Active EnZyme family 6 glycosyltransferases but also within both retaining and inverting GT-A fold glycosyltransferases. Our findings augment recently published crystal structures, which have identified a correlation between donor substrate conformational changes and mobile loop ordering.

The metabolism of glycosphingolipids by the malaria-causing parasite Plasmodium falciparum plays an important role in the progression of the disease. We report a new and highly sensitive method to monitor the uptake of glycosphingolipids in infected red blood cells (iRBCs). A tetramethylrhodamine-labeled glycosphingolipid (GM1TMR) was used as a substrate. Uptake was demonstrated by fluorescence microscopy. The iRBCs were lysed with a 15% solution of saponin and washed with phosphate buffered saline to release intact parasites. The parasites were further lysed and the resulting homogenates were analyzed by capillary electrophoresis with laser-induced fluorescence detection. The lysate from erythrocytes infected at 1% parasitemia generated a signal 20 standard deviations larger than uninfected erythrocytes, which suggests that relatively low infection levels can be studied with this technique. 10.1021/ac1021776

Glycosphingolipids are found on all vertebrate cells and constitute major cell surface determinants on all nerve cells, where they contribute to cellular diversity and function. We report a method for the analysis of glycosphingolipid metabolism in single cells. The ganglioside [G.sub.M1] was tagged with the fluorescent dye tetramethylrhodamine. This labeled compound was taken up and metabolized by a culture of pituitary tumor (AtT-20) cells. After 50 h, the cells were formalin fixed. Cells were aspirated into a fused-silica capillary and lysed, and components were separated by capillary electrophoresis with a laser-induced fluorescence detector. All metabolic products that retained the fluorescent dye could be detected at the low-zeptomole level. A total of 54 AtT-20 cells were individually analyzed using this procedure. The electrophoretic profiles were remarkably reproducible, which facilitated identification of components based on the migration time of fluorescently labeled standards. Eleven components were detected, and the average peak height of these components spanned more than 2 orders of magnitude, so that trace metabolites can be detected in the presence of abundant components. The most highly abundant components generated 10% relative standard deviation in normalized abundance. The average cell took up roughly 2 amol ([10.sup.6] copies) of the labeled substrate. This method allows determination of cell-to-cell diversity and regulation of glycosphingolipid metabolism.

The biosynthesis of human blood group B antigens is accomplished by a highly specific galactosyltransferase (GTB). On the basis of NMR experiments, a molecular tweezers mechanism that accounts for the exquisite stereoselectivity of donor substrate selection is proposed. Transferred NOE experiments reveal the bioactive conformation of the donor substrate UDP-galactose (UDP-Gal) and of its enzymatically inactive analogue, UDP-glucose (UDP-Glc).

The synthesis of a trisaccharide fragment (1) of the lipoarabinomannan from Tsukamurella paurometabola is reported. Two approaches were investigated for the synthesis of the target. One was purely chemical, while the other involved the addition of one of the monosaccharide residues via a mannosyltransferase-catalyzed reaction. Both approaches produced the target in good overall yields. Thus, this chemoenzymatic approach appears to be a useful addition to the arsenal of methods for the synthesis of lipoarabinomannan-derived oligosaccharides. Key words: lipoarabinomannan, oligosaccharide, mannosyltransferase, enzymatic synthesis. On a realise la synthese d'un fragment trisaccharide (1) du lipoarabinomannane extrait du Tsukamurella paurometabola. On a etudie deux approches pour la synthese de ce produit cible. L'une est purement chimique alors que l'autre implique l'addition des residus monosaccharides par le biais d'une reaction catalysee par la mannosyltransferase. Les deux approches ont permis d'arriver a la cible avec de bons rendements globaux. L'approche chimioenzymatique semble donc etre une addition utile a l'arsenal des methodes de synthese des oligosaccharides derives du lipoarabinomannane. Mots cles : lipoarabinomannane, oligosaccharide, mannosyltransferase, synthese enzymatique. [Traduit par la Redaction], Introduction The human pathogen Mycobacterium tuberculosis produces an immunomodulatory lipidated polysaccharide, lipoarabinomannan (LAM), which plays a critical role in the progression of tuberculosis (1, 2). This disease, and those caused [...]

This work presents new frontal affinity chromatography (FAC) methodologies for high-throughput screening of compound libraries, designed to increase screening rates and improve sensitivity and ruggedness in performance. A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented in the identification of potential enzyme inhibitors from two libraries of trisaccharides. Effluent from the FAC column was fractionated, sequentially processed via LC/MS, and referenced to a similar analysis through a control FAC column lacking the enzyme. The resulting multidimensional data sets were compared across corresponding sample and control fractions to identify binders, in a semiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first library of 81 compounds, exhibiting an estimated [K.sub.d] value of 0.3 [micro]M. Other binders yielded [K.sub.d] values ranging from 0.35 to 3.35 [micro]M. To demonstrate the improvement in performance of this FAC-LC/MS approach over the conventional online FAC/MS approach, 15 compounds from this library were blended with a second library of 1000 synthetic trisaccharides and screened against GnT-V. All ligands in the 15-compound set were identified in this larger screen, and no ligands of greater affinity than compound 1 were found. Our results show that FAC-LC/MS is a reliable method for screening large compound libraries directly and useful for large-scale ligand discovery initiatives.

A novel method for assaying enzymes from a single cell or small cell populations is described. The key advantage of this method is the ability to repeatedly sample a single cell enzyme reaction. Whereas multiple sampling has been achieved for larger cell types with a diameter of 1 mm, we report a technique by which single cell enzyme assays of small cells (15 [micro]m in diameter) can be repeatedly carried out. Individual cells were isolated using an in-house-built micromanipulator and placed in nanoliter-scale reaction vessels. The cells were lysed with solution containing substrate, and enzyme activity was assayed by removing 5-nL aliquots with a recently developed nanopipettor. The reaction aliquot was then analyzed using capillary electrophoresis with laser-induced fluorescence detection to quantitate enzyme activity. Sf9 cells were assayed at the single cell level and found to be highly heterogeneous with respect to [alpha]-glucosidase II activity. Since only 5 nL of the single cell reaction was removed, multiple sampling was possible, allowing triplicate analysis of enzyme activity for each individual cell. Multiple sampling also permitted a single cell reaction to be monitored over time. The sensitivity of this method was demonstrated in the analysis of a low-abundance enzyme, [alpha]1,3-N-acetylgalactosaminyltransferase, from single HT29 cells. Detecting the product of this enzyme reaction required minimizing the dilution of cellular contents. To demonstrate the potential applications of this methodology in small scale biochemical analyses, single Arabidopsis knf embryos lacking the [alpha]-glucosidase I encoding KNOPF gene were assayed. Mutant embryos demonstrated insignificant conversion of a triglucose substrate, as compared to wild type, confirming the deletion of [alpha]-glucosidase I. Embryos were simultaneously assayed for a second enzyme, [beta]-galactosidase, illustrating that the mutants were viable except for their lack of [beta]-glucosidase I activity.

A novel and readily available pipettor capable of nanolitersized volume manipulation was developed to improve and increase the flexibility of small-scale reaction processing. The volume delivery was found to be reproducible, with typical relative standard deviations of 1-5%, and easily tunable over a range of nanoliter-sized aliquots. The nanopipettor was combined with capillary electrophoresis using laser-induced fluorescence detection to monitor a small-scale enzyme reaction ([beta]-galactosidase) using a tetramethylrhodamine-labeled substrate. The results were in good agreement with a standard enzyme assay using a micropipet, thus demonstrating the nanopipettor's potential in developing new nanoscale utrasensitive enzyme assays.

The enzymatic synthesis of trimeric sialyl [Lewis.sup.x] pentadecasaccharide (6), a 15-mer, from a trimannoside precursor required six different glycosyltransferase enzymes and four nucleotide donor sugars. Three N-acetylglucosaminyl residues were transferred from UDP-N-acetylglucosamine to a trimannoside by N-acetylglucosaminyltransferases I, II, and V, respectively. Galactosylation using [beta](1 [right arrow] 4) galactosyltransferase and UDP-galactose gave three N-acetyllactosamine units in nonasaccharide 4. Sialylation of 4 with [alpha](2 [right arrow] 3) sialyltransferase and CMP-N-acetylneuraminic acid was followed by fucosylation with [alpha](1 [right arrow] 3) fucosyltransferase and GDP-fucose giving the 15-mer 6 in mg quantities. Compound 4 was also converted to a trimeric [Lewis.sup.x] dodecasaccharide 12-mer with [alpha](1 [right arrow] 3) fucosyltransferase and GDP-fucose and to a trimeric [alpha] -2,6-sialyl N-acetyllactosamine dodecasaccharide 12-mer with [alpha](2 [right arrow] 6) sialyltransferase and CMP-N-acetylneuraminic acid. Key words: glycosyltransferases, pentadecasaccharide, sialyl [Lewis.sup.x.]

Novel mutations in the RSW1 and KNOPF genes were identified in a large-scale screen for mutations that affect cell expansion in early Arabidopsis embryos. Embryos from both types of mutants were radially swollen with greatly reduced levels of crystalline cellulose, the principal structural component of the cell wall. Because RSW1 was previously shown to encode a catalytic subunit of cellulose synthase, the similar morphology of knf and rsw1-2 embryos suggests that the radially swollen phenotype of knf mutants is largely due to their cellulose deficiency. Map-based cloning of the KNF gene and enzyme assays of knfembryos demonstrated that KNF encodes [alpha]-glucosidase I, the enzyme that catalyzes the first step in N-linked glycan processing. The strongly reduced cellulose content of knf mutants indicates that N-linked glycans are required for cellulose biosynthesis. Because cellulose synthase catalytic subunits do not appear to be N glycosylated, the N-glycan requirement apparently resides in other component(s) of the cellulose synthase machinery. Remarkably, cellular processes other than extracellular matrix biosynthesis and the formation of protein storage vacuoles appear unaffected in knfembryos. Thus in Arabidopsis cells, like yeast, N-glycan trimming is apparently required for the function of only a small subset of N-glycoproteins.

Deoxygenated uridine 5'-diphosphogalactos derivatives were evaluated as donors for retaining galactosyltransferas.

Capillary electrophoresis is ideally suited to chemical analysis of individual cells. Small mammalian somatic cells (approximately 15 (mu)m in diameter) can be analyzed by injecting the intact cell into a capillary, lysing the cell, separating and detecting the cellular components, and reconditioning the capillary prior to the next injection. In this paper, we report on technical improvements to single-cell analysis. We designed an inexpensive multipurpose single-cell injector that facilitates the following: (i) monitoring of injection, (ii) reproducible pressure- or electrokinetic-driven injection of the cell, (iii) complete cell lysis by SDS within 30 s of injection, and (iv) pressure-driven capillary reconditioning. Furthermore, we report on the analysis of glycosylation and glycolysis in single human carcinoma cells (HT29 cell line). The reliability and quality of the analysis is confirmed by comparing electropherograms from single cells and those from purified cell extracts.

Research was conducted to express, characterize and compare alpha1,2-fucosyltransferase of Helicobacter pylori with the mammalian type. Results show the enzyme can be overproduced in Escherichia coli, is involved in the synthesis of Lewis antigens of lipopolysaccharide and differs from mammalian enzyme in several properties.