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Biomedical subjects

M M Palcic

Publications and source records attributed to M M Palcic.

At least 37 records · Page 2Linked to original sources
PubMed2000

Synthesis of mono- and di-fucosylated type I Lewis blood group antigens by Helicobacter pylori.

The identification of Helicobacter pylori isolates that expresses exclusively type I Lewis antigens is necessary to determine the biosynthetic pathway of these antigens. Fast-atom bombardment MS provides evidence that the H. pylori isolate UA1111 expresses predominantly Leb, with H type I and Lea in lesser amounts. Cloning and expression of the H. pylori fucosyltransferases (FucTs) allow comparisons with previously identified H. pylori enzymes and determination of the enzyme specificities. Although all FucTs, one alpha(1,2) FucT and two alpha(1,3/4) FucTs, appear to be functional in this isolate, their activities are lower and enzyme specificities are different to other H. pylori FucTs previously characterized. Studies of the cloned enzyme activities and mutational analysis indicate that Lea acts as the substrate for the synthesis of Leb. This is different from the human Leb biosynthetic pathway, but analogous to the biosynthetic pathway utilized by H. pylori for the production of Ley.

Amino Acid Sequence↗
PubMed2000

A novel viral alpha2,3-sialyltransferase (v-ST3Gal I): transfer of sialic acid to fucosylated acceptors.

The substrate specificity of an alpha2,3-sialyltransferase (v-ST3Gal I) obtained from myxoma virus infected RK13 cells has been determined. Like mammalian sialyltransferase enzymes, the viral enzyme contains the characteristic L- and S-sialyl motif sequences in its catalytic domain. Analysis of the deduced amino acid sequences of cloned sialyltransferases suggests that v-ST3Gal I is closely related to mammalian ST3Gal IV. v-ST3Gal I catalyzes the transfer of sialic acid from CMP-NeuAc to Type I (Galbeta1-3GlcNAcbeta) II (Galbeta1-4GlcNAcbeta) and III (Galbeta1-3GalNAcbeta) acceptors. In addition, the viral enzyme also transfers sialic acid to the fucosylated acceptors Lewis(x) and Lewis(a). This substrate specificity is unlike any sialyltransferases described to date, though it is most comparable with those of mammalian ST3Gal IV enzymes. The products from reactions with fucosylated acceptors were characterized by capillary zone electrophoresis, (1)H-NMR spectroscopy and mass spectrometry. They were shown to be 2,3-sialylated Lewis(x) and 2,3-sialylated Lewis(a), respectively.

Animals↗
PubMed1999

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.

Biodegradation, Environmental↗
PubMed1999

Biocatalytic synthesis of oligosaccharides.

Tremendous advances in biocatalytic approaches to oligosaccharide synthesis have taken place in the past two years. The use of isolated enzymes, both glycosyltransferases and glycosidases, or engineered whole cells allows the preparation of natural oligosaccharides and analogs required for glycobiology research.

Biotechnology↗
PubMed1999

Synthesis of the acceptor analog alphaFuc(1-->2)alphaGal-O(CH2)7 CH3: a probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase.

We report the chemical synthesis of alphaFuc(1-->2)alphaGal-O(CH2)7CH3 (1) an analog of the natural blood group (O)H disaccharide alphaFuc(1-->2)betaGal-OR. Compound 1 was a good substrate for recombinant blood group B glycosyltransferase (GTB) and was used as a precursor for the enzymatic synthesis of the blood group B analog (alphaGal(-->3)alphaFuc(1-->2)]alphaGal-O(CH2)7CH3+ ++ (2). To probe the mechanism of the GTB reaction, kinetic evaluations were carried out employing compound 1 or the natural acceptor disaccharide alphaFuc(1-->2)betaGal-O(CH2)7CH3 (3) with UDP-Gal and UDP-GalNAc donors. Comparisons of the kinetic constants for alternative donor and acceptor pairs suggest that the GTB mechanism is Theorell-Chance where donor binding precedes acceptor binding. GTB operates with retention of configuration at the anomeric center of the donor. Retaining reactions are thought to occur via a double-displacement mechanism with formation of a glycosyl-enzyme intermediate consistent with the proposed Theorell-Chance mechanism.

ABO Blood-Group System↗
PubMed1999

Processing alpha-glucosidase I is an inverting glycosidase.

Alpha-glucosidase I is a key enzyme in the biosynthesis of asparagine-linked oligosaccharides catalyzing the first processing event after the en bloc transfer of Glc3Man9GlcNAc2 to proteins. This enzyme is an inhibitor target for anti-viral agents that interfere with the formation of essential glycoproteins required in viral assembly, secretion and infectivity. Of fundamental mechanistic interest for all oligosaccharide hydrolyzing enzymes is the stereochemical course of the reaction which can occur with either retention or inversion of anomeric configuration. The stereochemistry is used to categorize enzymes and is important in designing mechanism-based inhibitors. To determine the stereochemical course of the alpha-glucosidase I reaction, the release of glucose from a synthetic trisaccharide substrate, Glc(alpha1-2)Glc(alpha1-3)Glc alphaO(CH2)8COOCH3 was directly monitored by 1H NMR spectroscopy. Both the yeast and bovine mammary gland enzymes released beta-glucose concomitant with the formation of the Glc(alpha1-3)Glc alphaO(CH2)8COOCH3 disaccharide product demonstrating that both enzymes operate with inversion of anomeric configuration.

Animals↗
PubMed1999

Donor substrate specificity of recombinant human blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli.

The human blood group A and B glycosyltransferases catalyze the transfer of GalNAc and Gal, to the (O)H-precursor structure Fuc alpha (1-2)Gal beta-OR to form the blood group A and B antigens, respectively. Changing four amino acids (176, 235, 266 and 268) alters the specificity from an A to a B glycosyltransferase. A series of hybrid blood group A/B glycosyltransferases were produced by interchanging these four amino acids in synthetic genes coding for soluble forms of the enzymes and expressed in Escherichia coli. The purified hybrid glycosyltransferases were characterized by two-substrate enzyme kinetic analysis using both UDP-GalNAc and UDP-Gal donor substrates. The A and B glycosyltransferases were screened with other donor substrates and found to also utilize the unnatural donors UDP-GlcNAc and UDP-Glc, respectively. The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs. B donor specificity.

Carbohydrate Sequence↗
PubMed1999

Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast.

Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-->2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate.

Cytological Techniques↗
PubMed1999

Molecular cloning, expression and exon/intron organization of the bovine beta-galactoside alpha2,6-sialyltransferase gene.

In this study, we report the first isolation and characterization of a bovine sialyltransferase gene. Bovine cDNAs prepared from different tissues contain an open-reading frame encoding a 405 amino acid sequence showing 83%, 75%, and 60% identity with human, murine, and chicken ST6Gal I (beta-galactoside alpha2,6-sialyltransferase) sequences, respectively. When transfected into COS-7 cells, a recombinant enzyme was obtained which catalyzed the in vitro alpha2, 6-sialylation of LacNAc (NeuAcalpha2-6Galbeta1-4GlcNAc) and LacdiNAc (NeuAcalpha2-6GalNAcbeta1-4GlcNAc) acceptor substrates. The K (m) values were 2.8 and 6.9 mM, respectively. Different relative efficiencies (Vmax/Km) for the two precursors (36 for LacNAc and 4.3 for LacdiNAc) were observed. Bovine ST6Gal I gene consists of four 5'-untranslated exons E(-2) to E(1), and five coding exons from E(2) to E(6). This later carries a 3'-untranslated region of 2. 7 kb. Gene sequence spans at least 80 kb of genomic DNA. Two processed pseudogenes have been identified. They are 94.3 and 95.6% similar to the bovine cDNA, respectively. Three families of mRNA isoforms were isolated. They differed by their 5'-untranslated regions and could be generated by three tissue-specific promoters. Family 1 is made up of exons E(-2) and E(1) to E(6), family 2 of exons E(-1) to E(6), and family 3 of exons E(1) to E(6). Tissular distribution of transcript families appears noticeably different than those described in human and rat.

5' Untranslated Regions↗
PubMed1998

Identification of the quinone cofactor in mammalian semicarbazide-sensitive amine oxidase.

Mammalian semicarbazide-sensitive amine oxidase (SSAO) enzymes have been classified as EC 1.4.3.6 [amine:oxygen oxidoreductase (deaminating)(copper-containing)]. However, both the identity of the quinone cofactor and the presence of copper remain unconfirmed, and SSAO has proved impossible to purify to homogeneity in sufficient yield to permit cofactor identification. To circumvent this problem, we have partially purified SSAO enzymes from bovine and porcine aortae and have established, with a redox-cycling assay, that no other quinoproteins were present in enzyme preparations. Enzymes were then derivatized with (p-nitrophenyl)hydrazine (p-NPH), which forms a covalent yellow complex with the quinone cofactor. Visible absorbance spectra of derivatized bovine and porcine enzymes (respective lambdamax values 456 and 476 nm at neutral pH, shifting to 580 and 584 nm in 2 M KOH) were consistent with the presence of (2,4,5-trihydroxyphenyl)alanine quinone (TPQ) as cofactor. Resonance Raman spectra were essentially identical to that for pea seedling amine oxidase, a known TPQ-containing enzyme. Extensive digestion of SSAO enzymes, and of porcine kidney diamine oxidase, with pronase E yielded species with identical chromophoric properties characteristic of the dipeptide, TPQ(p-NPH)-Asp. Thermolytic digestion of porcine SSAO gave two cofactor-containing peptides that contained a TPQ consensus sequence, Asn-X-Asp-Tyr-Tyr, where X is a blank cycle corresponding to TPQ. N-terminal sequencing of whole enzymes revealed a membrane-spanning region typical of an extracellular type II glycoprotein. These results confirm the presence of TPQ in mammalian membrane-bound SSAO ectoenzymes.

Amine Oxidase (Copper-Containing)↗
PubMed1998

UDP-N-trifluoroacetylglucosamine as an alternative substrate in N-acetylglucosaminyltransferase reactions.

The synthesis of UDP-N-trifluoroacetylglucosamine [uridine 5'-(2-trifluoroacetamido-2-deoxy-alpha-D-glucopyranosyl diphosphate, UDP-GlcNAc-F3] is reported. The compound is found to serve as a substrate for the core-2 GlcNAc transferase (EC 2.4.1.102) that is involved in the biosynthesis of O-linked glycoproteins and for the GlcNAcT-V transferase (EC 2.4.1.155) that is a key biosynthetic enzyme controlling the branching pattern of cell surface complex Asn-linked oligosaccharides. The trisaccharide beta-D-Galp-(1-->3) -[beta-D-GlcpNAc-F3(1-->6)] alpha-D-GalpNAc-OR [R = (CH2)8CO2Me] was prepared from beta-D-Galp-(1-->3) -alpha-D- GalpNAc-OR using the core-2 GlcNAc transferase. The tetrasaccharide beta-D-GlcpNAc-(1 -->2)-[beta-D-GlcpNAc-F3-(1-->6)]-alpha-D-Manp-(1-->6)-beta-D-Glcp -OR [R = (CH2)7CH3] using the GlcNAcT-V transferase. Removal of the trifluoroacetyl group was achieved under mild basic conditions to give the corresponding glucosamine containing tetrasaccharide. These examples demonstrate the feasibility of introducing masked forms of glucosamine residues into oligosaccharides using GlcNAc-specific transferases. The requirement for the trifluoroacetamido group as a specific recognition element was evident in the observation that neither UDP-glucosamine nor UDP-glucose served as a donor substrates for the core-2 GlcNAc transferase.

Acetylglucosamine↗
PubMed1997

Synthesis and evaluation of eight aminodeoxy trisaccharide inhibitors for N-acetylglucosaminyltransferase-V.

N-Acetylglucosaminyltransferase-V is an important enzyme controlling the branching pattern of N-linked oligosaccharides. This enzyme recognizes the trisaccharide octyl 2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1-->2)-alpha-D-mannopyranosyl -(1-->6)-beta-D-glucopyranoside (5) as a substrate and adds a beta-linked GlcNAc residue to OH-6 of the central alpha-Man unit. Eight analogs of 5 were chemically synthesized where C-6 of the alpha-Man residue in 5 was deoxygenated, and structurally diverse modifications were introduced at C-4 of the same residue. The key intermediate prepared for this purpose was octyl 2-acetamido-2-deoxy-beta-D- glucopyranosyl-(1-->2)-4-amino-4,6-dideoxy-alpha-D-mannopyranosyl- (1-->6)-beta-D-glucopyranoside (7a) where the original 4'-amino group was readily derivatized on the unprotected sugar. The eight analogs 7a-7h were evaluated as inhibitors for GlcNAcT-V, both isolated (from hamster kidney) and cloned (from rat kidney). All of the compounds were found to be competitive inhibitors with Ki in the range of 3-106 microM. The conclusion of this work is that recognition of acceptor 5 does not involve contact of the C-6--C-4 end of the alpha-Man residue with the protein in the E-I (or E-S) complex.

Amino Sugars↗
PubMed1997

Study of the enzymatic transformation of fluorescently labeled oligosaccharides in human epidermoid cells using capillary electrophoresis with laser-induced fluorescence detection.

Isomeric oligosaccharides of both beta Gal(1-->3)beta GlcNAc (type I) series and beta Gal(1-->4)beta GlcNAc (type II) series were studied by using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. A mixture of phenylboronic acid and sodium tetraborate was used in the CE running buffers to improve the electrophoretic separation of the oligosaccharides. Both series of the tetramethylrhodamine (TMR)-labeled substrates [beta Gal(1-->3)beta GlcNAc-O-TMR and beta Gal(1-->4)beta GlcNAc-O-TMR) and their potential enzymatic products were baseline resolved using CE. The high resolution provided by CE and the excellent detection limit (8.10(-23) mol, or 50 molecules) by LIF allowed for the determination of minor enzyme products in the presence of excess unreacted substrate. The action of competing enzymes acting on the common type I sequence was monitored after the incubation of human epidermoid carcinoma cells (A431) with a fluorescent substrate (beta Gal(1-->3)beta GlcNAc-O-TMR). The CE-LIF analyses showed the formation of both synthetic and hydrolytic products, suggesting the actions of glycosyltransferases and glycosidases in the cells.

Carbohydrate Sequence↗
PubMed1997

Cloning and heterologous expression of an alpha1,3-fucosyltransferase gene from the gastric pathogen Helicobacter 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 alpha1,3-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 alpha1,3/1,4-fucosyltransferases. The approximately 52-kDa protein encoded by HpfucT was expressed in Escherichia coli CSRDE3 cells and gave rise to alpha1,3-fucosyltransferase activity but neither alpha1,4-fucosyltransferase nor alpha1,2-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 alpha1,3-fucosyltransferases. These lines of evidence indicate that the HpFuc-T represents the bacterial alpha1,3-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 alpha1,3-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.

Amino Acid Sequence↗
PubMed1997

Chemoenzymatic synthesis of a trimeric ganglioside GM3 analogue.

A trimeric beta-lactosyl cluster based on 2-nitro-2-(hydroxymethyl)propane-1,3-diol was prepared using the trichloroacetimidate method. Kinetic studies showed that this cluster was an effective acceptor for rat-liver alpha-(2-->3)-sialyltransferase. Its KM was comparable to those for monomeric lactose and N-acetyllactosamine acceptors, and its Vmax was 1% of that measured for the LacNAc acceptor. Preparative-scale sialylation using this enzyme afforded a trimeric cluster of the ganglioside GM3 oligosaccharide in good yield. The NMR spectra of the trimeric GM3 analogue suggest that the oligosaccharide conformation is not significantly perturbed by this level of clustering.

Amino Sugars↗
PubMed1997

Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes.

The human blood group A and B glycosyltransferase enzymes are highly homologous and the alteration of four critical amino acid residues (Arg-176 --> Gly, Gly-235 --> Ser, Leu-266 --> Met, and Gly-268 --> Ala) is sufficient to change the enzyme specificity from a blood group A to a blood group B glycosyltransferase. To carry out a systematic study, a synthetic gene strategy was employed to obtain their genes and to allow facile mutagenesis. Soluble forms of a recombinant glycosyltransferase A and a set of hybrid glycosyltransferase A and B mutants were expressed in Escherichia coli in high yields, which allowed them to be kinetically characterized extensively for the first time. A functional hybrid A/B mutant enzyme was able to catalyze both A and B reactions, with the kcat being 5-fold higher for the A donor. Surprisingly, even a single amino acid replacement in glycosyltransferase A with the corresponding residue from glycosyltransferase B (Arg-176 --> Gly) produced enzymes with glycosyltransferase A activity only, but with very large (11-fold) increases in the kcat and increased specificity. The increases observed in kcat are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synthesis of blood group antigens.

ABO Blood-Group System↗