Preliminary joint neutron time-of-flight and X-ray crystallographic study of human ABO(H) blood group A glycosyltransferase.

The biosyntheses of oligosaccharides and glycoconjugates are conducted by glycosyltransferases. These extraordinarily diverse and widespread enzymes catalyze the formation of glycosidic bonds through the transfer of a monosaccharide from a donor molecule to an acceptor molecule, with the stereochemistry about the anomeric carbon being either inverted or retained. Human ABO(H) blood group A α-1,3-N-acetylgalactosaminyltransferase (GTA) generates the corresponding antigen by the transfer of N-acetylgalactosamine from UDP-GalNAc to the blood group H antigen. To understand better how specific active-site-residue protons and hydrogen-bonding patterns affect substrate recognition and catalysis, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection and time-of-flight neutron diffraction data were collected to 2.5 Šresolution at the PCS to ∼85% overall completeness, with complementary X-ray diffraction data collected from a crystal from the same drop and extending to 1.85 Šresolution. Here, the first successful neutron data collection from a glycosyltransferase is reported.

Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583.

AIMS: To compare enzymatic activities of two related chitinases, ChiA and EF0361, encoded by Listeria monocytogenes and Enterococcus faecalis, respectively. METHODS AND RESULTS: The chiA and EF0361 genes were amplified by PCR, cloned and expressed with histidine tags, allowing easy purification of the gene products. ChiA had a molecular weight as predicted from the amino acid sequence, whereas EF0361 was 1840 Da lower than expected because of C-terminal truncation. The ChiA and EF0361 enzymes showed activity towards 4-nitrophenyl N,N'-diacetyl-beta-D-chitobioside with K(m) values of 1.6 and 2.1 mmol l(-1), respectively, and k(cat) values of 21.6 and 6.5 s(-1). The enzymes also showed activity towards 4-nitrophenyl beta-D-N, N', N''-triacetylchitotriose and carboxy-methyl-chitin-Remazol Brilliant Violet but not towards 4-nitrophenyl N-acetyl-beta-D-glucosaminide. Chitinolytic specificities of the enzymes were supported by their inactivity towards the substrates 4-nitrophenyl beta-D-cellobioside and peptidoglycan. The pH and temperature profiles for catalytic activities were relatively similar for both the enzymes. CONCLUSION: The ChiA and EF0361 enzymes show a high degree of similarity in their catalytic activities although their hosts share environmental preferences only to some extent. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to an understanding of the chitinolytic activities by L. monocytogenes and Ent. faecalis. Detailed information on their chitinolytic systems will help define potential reservoirs in the natural environment and possible transmission routes into food-manufacturing plants.

The proteolysis-inducing factor: in search of its clinical relevance in patients with metastatic gastric/esophageal cancer.

The proteolysis-inducing factor is a putative mediator of cancer-associated weight loss. The goal of this study was to examine for the first time: (i) its prevalence in patients with metastatic gastric/esophageal cancer; and (ii) whether it possibly correlated with weight loss and anorexia and whether it predicted tumor response and patient survival. This study recruited 41 patients as part of a phase II therapeutic, chemotherapy protocol for patients with metastatic gastric/esophageal cancer. Patient eligibility criteria were designed to select a group of patients who would tolerate treatment with the drugs capecitabine and oxaliplatin. Urine for assaying the proteolysis-inducing factor was obtained at registration and then 6 weeks later. Patients completed the FACT-E questionnaire every 6 weeks and had their weights checked at the same interval. Patients were followed prospectively for tumor response and patient survival. Twenty-three (56%) patients had the proteolysis-inducing factor in their urine at registration, and 18 (64%) had it at 6 weeks. There was no statistically significant correlation between the presence of the proteolysis-inducing factor and weight loss or between its presence and anorexia. Moreover, there was no evidence that the presence of the proteolysis-inducing factor in urine was able to predict tumor response or patient survival. The proteolysis-inducing factor in urine does not appear to be tied to weight loss, anorexia, tumor response, or patient survival in the clinical setting of metastatic gastric/esophageal cancer.

Kinetic studies on Korean serum cis-AB enzymes reveal diminished A and B transferase activities.

BACKGROUND: cis-AB enzymes are rare glycosyltransferases that synthesize both blood group A and B antigens. We have identified a large cohort of Korean cis-AB blood donors and studied the N-acetylgalactosaminyltransferase (glycosyltransferase A, GTA) and galactosyltransferase (glycosyltransferase B, GTB) activity of their cis-AB serum enzymes. MATERIALS AND METHODS: The cis-AB01 allele was identified by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) in 60 donors collected at the Gwangju-Chonnam Red Cross Blood Center. Enzyme assays of this cis-AB enzyme were performed on available serum samples from 16 donors with the cis-AB01/O genotype and three with the cis-AB01/A genotype. RESULTS: In cis-AB donors with an O allele, both the GTA and GTB activity of the cis-AB enzyme were markedly reduced compared to normal A and B controls (29% and 27%, respectively). This is consistent with the behaviour predicted from kinetic studies of a recombinant model of the corresponding AAAB enzyme. CONCLUSION: Although variable, cis-AB enzymes feature reduced GTA and GTB activities. SUMMARY: Cis-AB enzymes feature variable but reduced GTA and GTB activities with relatively weaker GTB activity, consistent with the weak agglutination present on forward typing with anti-B.

An evaluation of the activation of endogenous pancreatic enzymes during human islet isolations.

Despite advances in human islet isolations, there remain inconsistencies in human islet yield and viability after collagenase digestion. It has been suggested that trypsin may contribute to the proteolysis of collagenase and the destruction of islet cells, or possibly exert indirect effects on the pancreas by activating other endogenous serine proenzymes. This study evaluated the effects of serine proteases on collagenase activity and profiled the kinetics of serine protease activity throughout human islet isolations with and without addition of Pefabloc, a serine protease inhibitor. Cadaveric pancreases were perfused in the presence (n = 12) and absence of Pefabloc (0.4 mmole; n = 8). Samples were collected before and throughout the digestion process and were assayed for trypsin, chymotrypsin, and elastase activity. A study of the enzyme kinetics of serine proteases throughout human islet isolations showed an increase in activity levels throughout the digestion period. There was a significant difference in the chymotrypsin (1342 +/- 503 and 384 +/- 71 units) and elastase (7.94 +/- 1.1 and 2.761 +/- 0.69 units) levels between the control and Pefabloc-supplemented isolations, respectively. There was no significance difference noted among the trypsin (88 +/- 27 and 54 +/- 18 units) levels between the control and Pefabloc-supplemented isolations, respectively. This demonstrates that serine proteases are effectively inhibited by Pefabloc during the islet isolation process. These data show that the presence of serine proteases may likely damage the islets upon prolonged digestion of the pancreatic tissue.

An evaluation of endogenous pancreatic enzyme levels following enzymatic digestion.

Human islet isolation consists of a digestion phase, dilution phase, and purification phase. Recent evidence suggests that inconsistencies in islet yields are attributed to the activation of endogenous enzymes of the donor pancreas during the digestion phase. Therefore, following the digestion phase, it is important to inhibit these enzymes by the addition of an inhibitor to the dilution phase. In this study, we report the endogenous pancreatic enzyme levels after the purification phase and the effects of potential inhibitors on the proteases of interest. Results at the end of the purification phase indicated that chymotrypsin retained approximately 20% of the activity observed after the digestion phase, whereas trypsin, elastase, and collagenase retained approximately 2.5%, 2%, and 3.5% of the activity, respectively, demonstrating that added inhibitors are not fully effective. Potential enzyme inhibitors, human albumin, fetal calf serum, and aprotinin, were incubated with trypsin, chymotrypsin, elastase, and collagenase and assayed for activity. Fetal calf serum and aprotinin showed strong inhibitory actions toward trypsin and chymotrypsin. Aprotinin completely inhibited the tryptic activity; however, it did not inhibit human chymotrypsin or elastase activity. Human albumin showed minimal inhibition and was shown to act as a competitive inhibitor. This study clearly demonstrates that low amounts of endogenous pancreatic enzymes remain active at the end of the human islet isolation procedure and that the added inhibitors at the dilution are not fully effective at inhibiting the enzymes.

Frontal affinity chromatography coupled to mass spectrometry for screening mixtures of enzyme inhibitors.

Frontal affinity chromatography coupled online to mass spectrometry (FAC/MS) has previously been used to estimate binding constants for individual protein ligands present in mixtures of compounds. In this study FAC/MS is used to determine enzyme substrate kinetic parameters and binding constants for enzyme inhibitors. Recombinant human N-acetylglucosaminyltransferase V was biotinylated and adsorbed onto immobilized streptavidin in a microcolumn (20 microL). The enzyme was shown to be catalytically competent transferring GlcNAc from the donor UDP-GlcNAc to beta-d-GlcpNAc-(1-->2)-alpha-d-Manp-(1-->6)-beta-d-Glcp-OR acceptor giving beta-d-GlcpNAc-(1-->2)-[beta-d-GlcpNAc-(1-->6)]-alpha-d-Manp-(1-->6)-beta-d-Glcp-OR as the reaction product. The kinetic parameters K(m) and V(max) for the immobilized enzyme could be determined by FAC/MS and were comparable to those measured in solution. Analysis of a mixture of eight trisaccharide analogs in a single run yielded K(d) values for each of the eight compounds ranging from 0.3 to 36 microM. These K(d) values were 2 to 10 times lower than the inhibition constants, K(I)'s, determined in solution using a standard radiochemical assay. However, the ranking order of K(d)'s was the same as the ranking of K(I) values. FAC/MS assays can therefore be employed for the rapid estimation of inhibitor K(d) values making it a valuable tool for enzyme inhibitor evaluations.

Natural and recombinant A and B gene encoded glycosyltransferases.

The human blood group A and B synthesizing enzymes are glycosyltransferases that catalyse the transfer of a monosaccharide residue from UDP-GalNAc and UDP-Gal donors, respectively, to alphaFuc1,2-Gal terminated blood group H acceptors. Extensive investigations of their substrate specificity and physical properties have been carried out since their initial discovery. These studies demonstrated a rigid specificity for the acceptor structure, crossover in donor specificity and immunological similarity along with chromatographic differences. Cloning of the enzymes has shown that they are highly homologous, differing in only four of their 354 amino acids. Changing the residues Arg176-->Gly, Gly235-->Ser, Leu266-->Met and Gly 268-->Ala converts the enzyme specificity from blood group A to blood group B glycosyltransferase. Structure function investigations have been carried out by systematic interchange and modification of these four critical amino acids. These studies have shown that donor specificity is attributed to the last two amino acids. Mutants have also been produced with greatly enhanced turnover rates as well as hybrid A/B enzymes that catalyse both reactions efficiently.

Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry.

The binding affinity and specificity of the mushroom Polyporus squamosus lectin has been determined by the recently developed method of frontal affinity chromatography coupled to electrospray mass spectrometry (FAC/MS). A micro-scale affinity column was prepared by immobilizing the lectin ( approximately 25 microg) onto porous glass beads in a tubing column (9.8 microl column volume). The column was then used to screen several oligosaccharide mixtures. The dissociation constants of 22 sialylated or sulfated oligosaccharides were evaluated against the immobilized lectin. The lectin was found to be highly specific for Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc containing oligosaccharides with K(d) values near 10 microM. The FAC/MS assay permits the rapid determination of the dissociation constants of ligands as well as a higher throughput screening of compound mixtures, making it a valuable tool for affinity studies, especially for testing large numbers of compounds.

Effects of amine modifiers on the separation of tetramethylrhodamine-labeled mono- and oligosaccharides by capillary zone electrophoresis.

In this work, nine tetramethylrhodamine (TMR) labeled isomeric oligosaccharide derivatives of betaGal(1 --> 4) betaGlcNAc-O-TMR were separated by capillary zone electrophoresis coupled with laser-induced fluorescence detection. Charged species were created in situ by complexation with borate and phenylborate. Micellar separation was achieved by addition of 10 mM sodium dodecylsulfate to the running buffer. We have investigated the effects of adding a homologous series of monoamine modifiers on the separation efficiency of these oligosaccharides. The separation was significantly improved in the presence of the organic modifiers methyl- and ethylamines, but worsened in the presence of propyl- and butylamines. Possible mechanisms of the amine additives are discussed.

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.

Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry.

Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.

Intracellular inhibition of blood group A glycosyltransferase.

We report the intracellular inhibition of blood group A N-acetylgalactosaminyltransferase in the human colorectal carcinoma cell line HT29 by 3-amino-3-deoxy-[Fucalpha(1-2)]Galbeta-O(CH2)7CH3. Inhibition was demonstrated with a novel capillary electrophoresis assay that monitored decreased intracellular conversion of fluorescently labelled Fucalpha(1-2)Gal-R acceptor to the corresponding A epitope, GalNAcalpha(1-3)[Fucalpha(1-2)]Galbeta-R. Growth of HT29 cells with either the amino-inhibitor or a competitive substrate, Fucalpha(1-2)Galbeta-O(CH2)7CH3, also resulted in decreased expression of blood group A determinants on cell-associated glycoproteins, as detected by immunoprecipitation analysis using A-specific monoclonal antibodies. Furthermore, exposure of these cells to the amino-inhibitor or competitive substrate resulted in significant reduction of cell-surface expression of blood group A determinants. As integrin alpha3beta1, a cell-surface receptor mediating cell-cell and cell-extracellular matrix interactions, was shown previously to be a major carrier of blood group A determinants on HT29 cells, the studies described herein highlight the potential usefulness of these compounds for elucidating the role of blood group A determinants in biological phenomena.

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.

Enzymatic synthesis of blood group A and B trisaccharide analogues.

Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations. The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates. We now report a unique donor specificity for each enzyme type. Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2 )7CH3 (4). Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2) 7CH3 (5). In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc. Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5.

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.

Instrumentation for chemical cytometry.

Capillary electrophoresis is ideally suited to chemical analysis of individual cells. Small mammalian somatic cells (approximately 15 microns 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.

Cloning and characterization of the alpha(1,3/4) fucosyltransferase of Helicobacter pylori.

The gastric pathogen Helicobacter pylori can express the histo blood group antigens, which are on the surface of many human cells. Most H. pylori strains express the type II carbohydrates, Lewis X and Y, whereas a small population express the type I carbohydrates, Lewis A and B. The expression of Lewis A and Lewis X, as in the case of H. pylori strain UA948, requires the addition of fucose in alpha1,4 and alpha1,3 linkages to type I or type II carbohydrate backbones, respectively. This work describes the cloning and characterization of a single H. pylori fucosyltransferase (FucT) enzyme, which has the ability to transfer fucose to both of the aforementioned linkages in a manner similar to the human fucosyltransferase V (Fuc-TV). Two homologous copies of the fucT gene have been identified in each of the genomes sequenced. The characteristic adenosine and cytosine tracts in the amino terminus and repeated regions in the carboxyl terminus are present in the DNA encoding the two UA948fucT genes, but these genes also contain differences when compared with previously identified H. pylori fucTs. The UA948fucTa gene encodes an approximately 52-kDa protein containing 475 amino acids, whereas UA948fucTb does not encode a full-length FucT protein. In vitro, UA948FucTa appears to add fucose with a greater than 5-fold preference for type II chains but still retains significant activity using type I acceptors. The addition of the fucose to the type II carbohydrate acceptors, by UA948FucTa, does not appear to be affected by fucosylation at other sites on the carbohydrate acceptor, but the rate of fucose transfer is affected by terminal fucosylation of type I acceptors. Through mutational analysis we demonstrate that only FucTa is active in this H. pylori isolate and that inactivation of this enzyme eliminates expression of all Lewis antigens.