Nora W. C. Chan

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patients urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.

Cloning and Heterologous Expression of an α1,3-Fucosyltransferase Gene from the Gastric PathogenHelicobacter pylori

Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is also associated with gastric cancer and lymphoma. This microorganism has been shown to express cell surface glycoconjugates including Lewis X (Lex) and Lewis Y. These bacterial oligosaccharides are structurally similar to tumor-associated carbohydrate antigens found in mammals. In this study, we report the cloning of a novel α1,3-fucosyltransferase gene (HpfucT) involved in the biosynthesis of Lex within H. pylori. The deduced amino acid sequence of HpfucT consists of 478 residues with the calculated molecular mass of 56,194 daltons, which is approximately 100 amino acids longer than known mammalian α1,3/1,4-fucosyltransferases. The ∼52-kDa protein encoded byHpfucT was expressed in Escherichia coli CSRDE3 cells and gave rise to α1,3-fucosyltransferase activity but neither α1,4-fucosyltransferase nor α1,2-fucosyltransferase activity as characterized by radiochemical assays and capillary zone electrophoresis. Truncation of the C-terminal 100 amino acids of HpFuc-T abolished the enzyme activity. An approximately 72-amino acid region of HpFuc-T exhibits significant sequence identity (40–45%) with the highly conserved C-terminal catalytic domain among known mammalian and chicken α1,3-fucosyltransferases. These lines of evidence indicate that the HpFuc-T represents the bacterial α1,3-fucosyltransferase. In addition, several structural features unique to HpFuc-T, including 10 direct repeats of seven amino acids and the lack of the transmembrane segment typical for known eukaryotic α1,3-fucosyltransferases, were revealed. Notably, the repeat region contains a leucine zipper motif previously demonstrated to be responsible for dimerization of various basic region-leucine zipper proteins, suggesting that the HpFuc-T protein could form dimers.

Correlating Cell Cycle With Metabolism in Single Cells: Combination of Image and Metabolic Cytometry

BACKGROUND We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.

Novel Helicobacter pylori α1,2-fucosyltransferase, a key enzyme in the synthesis of Lewis antigens

Helicobacter pylori lipopolysaccharides (LPS) contain complex carbohydrates known as Lewis antigens which may contribute to the pathogenesis and adaptation of the bacterium. Involved in the biosynthesis of Lewis antigens is an alpha1,2-fucosyltransferase (FucT) that adds fucose to the terminal betaGal unit of the O-chain of LPS. Recently, the H. pylori (Hp) alpha1,2-FucT-encoding gene (fucT2) was cloned and analysed in detail. However, due to the low level of expression and instability of the protein, its enzymic activity was not demonstrated. In this study, the Hp fucT2 gene was successfully overexpressed in Escherichia coli. Sufficient amounts of the protein were obtained which revealed alpha1,2-fucosyltransferase activity to be associated with the protein. A series of substrates were chosen to examine the acceptor specificity of Hp alpha1,2-FucT, and the enzyme reaction products were identified by capillary electrophoresis. In contrast to the normal mammalian alpha,2-FucT (H or Se enzyme), Hp alpha1,2-FucT prefers to use Lewis X [betaGal1-4(alphaFuc1-3)betaGlcNAc] rather than LacNAc [betaGal1-4betaGIcNAc] as a substrate, suggesting that H. pylori uses a novel pathway (via Lewis X) to synthesize Lewis Y. Hp alpha1,2-FucT also acts on type 1 acceptor [betaGal1-3betaGlcNAc] and Lewis a [betaGal1-3(alphaFuc1-4)betaGIcNAc], which provides H. pylori with the potential to synthesize H type 1 and Lewis b epitopes. The ability to transfer fucose to a monofucosylated substrate (Lewis X or Lewis a) makes Hp alpha1,2-FucT distinct from normal mammalian alpha1,2-FucT.

Frontal affinity chromatography-mass spectrometry assay technology for multiple stages of drug discovery: applications of a chromatographic biosensor.

This article presents new concepts in affinity chromatography/mass spectrometry for the study of molecular interactions. Chromatographic assays involving estrogen receptor-beta, sorbitol dehydrogenase, human alpha-thrombin, cholera toxin B subunit, beta-galactosidase, and Griffonia simplicifolia isolectin B(4) were established in microaffinity columns and operated in frontal analysis mode. Methods and formalism are presented for the measurement of dissociation constants, using direct methods in which the mass spectrometric signature of the ligand is used to measure breakthrough time and, hence, binding strength. The direct approach is capable of measuring sub-micromolar K(d) and higher, on sub-pmol amounts of immobilized protein, as shown in the cholera toxin assay. Indirect assays that demonstrate the advantage of routine, rugged performance were developed. By tracking the effect of a test ligand on a selected probe, or indicator ligand, dissociation constants in the low nanomolar range could be reliably determined for ligands to estrogen receptor-beta. Mass spectrometry supports the resolution of complex ligand mixtures, and it is demonstrated in the sorbitol dehydrogenase assay that ligands can be rank ordered across approximately three orders of magnitude in K(d), in a single run. A new concept for rapid mixture prescreening is presented, in which an indicator ligand can be used to discriminate between mixtures that contain high levels of weak ligands and those that contain single strong ligands.

Single-cell analysis avoids sample processing bias

Microscale separation tools such as capillary chromatography and capillary electrophoresis (CE) allow the study of metabolism in individual cells. In this work, we demonstrate that single-cell analysis describes metabolism more accurately than analysis of cellular extracts. We incubated HT29 cells (human colon adenocarcinoma) with a fluorescently labeled metabolic probe. This disaccharide, LacNAc, was labeled with a fluorescent dye, tetramethylrhodamine (TMR). The probe was taken up by the cells and metabolized to a number of products that retained the fluorescent label. We then split the cells into two batches. A cellular extract was prepared from one batch and analyzed by CE with laser-induced fluorescence (LIF) detection. The cells from the second batch were used for single-cell analysis by CE-LIF. Separation and detection conditions were identical for extract and single-cell analyses. We found that the electropherogram obtained by averaging the results from a number of single cells differed significantly from the cell extract electropherogram. Differences were due to sample processing during extract preparation. Disruption of the cells liberated enzymes that were compartmentalized within the cell, which allowed non-metabolic reactions to proceed. The accumulation of these non-metabolic products introduced a bias in the cell extract assay. During single-cell analysis, cells were lysed inside the capillary and the separation voltage was applied immediately to separate the enzymes from their substrates and prevent non-metabolic reactions. This paper is the first to report that CE analysis of single cells provides more accurate metabolic information than the CE analysis of a cellular extract.

Investigation of the Utility of Complementary Electrochemical Detection Techniques to Examine the in Vitro Affinity of Bacterial Flagellins for a Toll-Like Receptor 5 Biosensor

An initial investigation of the fabrication of a novel biosensor utilizing toll-like receptor 5 (TLR5) has been conducted. The detection assay using this sensor platform has been carried out using two complementary electrochemical techniques. The electrochemical properties of the modified bare gold surface following TLR5 immobilization were characterized. The electrochemical response to changes in the sensor film resistance and electron charge-transfer permittivity triggered by independent exposures to flagellins from Salmonella typhimurium (S. typhimurium) and Bacillus subtilis (B. subtilis) were examined and observed. The quantified film resistance data gathered using electrochemical impedance spectroscopy (EIS) over a macroscopic scale are in significant agreement with the corresponding electron charge-transfer permittivity measured locally by scanning electrochemical microscopy (SECM). Unlike other sensors that exploit pathogen recognition elements, TLR5 biosensors have the potential to carry out broad-spectrum detection of flagellated bacterial pathogens in near real time. This broad-spectrum detection platform is a significant step toward the development of fast, inexpensive clinical tools for early warning diagnoses and immediate on-site treatment.

Toll-like receptor 3 modified Au electrodes: an investigation into the interaction of TLR3 immobilized on Au surfaces with poly(I:C)

The rapid detection of viruses is crucial for medical diagnosis, environmental monitoring, public health, and homeland security. In this report, the development of a novel electrochemical impedance spectroscopy-based method utilizing Toll-Like Receptor 3 (TLR3)-modified Au electrodes for studying the interaction of TLR3 on a surface with polyinosinic–polycytidylic acid (poly(I:C)) as a dsRNA mimic (a molecular signature of viruses) has been described. The modified Au electrodes were prepared by covalent immobilization of TLR3 protein using lipoic acid N-hydroxysuccinimide ester (Lip-NHS) linkers. The electrochemical behavior of the modified electrodes and their applicability for sensing poly(I:C) was examined.

Characterization of TLR4/MD-2-modified Au sensor surfaces towards the detection of molecular signatures of bacteria

Lipopolysaccharides (LPSs), also known as endotoxins, can be fatal even at low concentrations. As a result, the development of novel methodologies for LPS detection has been continuously in the focus of research. Biosensors, which employ a bio-recognition element on a transducer surface, are on the cutting edge of these novel technologies. In this report, Au surfaces modified with TLR4/MD-2 through Lip-NHS linkers with an ultimate potential application as biosensors for LPS detection have been characterized and investigated using X-ray photoelectron spectroscopy, quartz crystal microbalance and electrochemical techniques. Also the interaction between TLR4/MD-2 immobilized on Au surfaces with LPSs has been studied to evaluate the possibility of LPS detection.