Multiple sampling in single-cell enzyme assays using capillary electrophoresis with laser-induced fluorescence detection.
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Animals↗
Biomedical subjects
Monica M Palcic
Publications and source records attributed to Monica M Palcic.
Blood group B galactosyltransferase: insights into substrate binding from NMR experiments.
The biosynthesis of human blood group B antigens is accomplished by a highly specific galactosyltransferase (GTB). On the basis of NMR experiments, we propose a "molecular tweezers mechanism" that accounts for the exquisite stereoselectivity of donor substrate selection. Transferred NOE experiments for the first time reveal the bioactive conformation of the donor substrate UDP-galactose (UDP-Gal) and of its enzymatically inactive analogue, UDP-glucose (UDP-Glc). Both bind to GTB in a folded conformation that is sparsely populated in solution, whereas acceptor ligands bind in a conformation that predominates in solution. The bound conformations of UDP-Gal and UDP-Glc are identical within experimental error. Therefore, GTB must discriminate between the two activated sugars on the basis of a hitherto unknown transition state that can only be formed in the case of UDP-Gal. A full relaxation and exchange matrix analysis of STD NMR experiments reveals that acceptor substrates dissociate significantly faster (k(off) > 100 Hz) from the binding pocket than donor substrates (k(off) approximately 10 Hz). STD NMR experiments also directly show that proper recognition of the hexopyranose rings of the UDP sugars requires bivalent metal cations. At the same time, this analysis furnishes the complete three-dimensional structure of the enzyme with its bound donor substrate UDP-Gal on the basis of a prior crystal structure analysis. We propose that, upon acceptor binding, GTB uses the Asp 302 and Glu 303 side chains as "molecular tweezers" to promote bound UDP-Gal but not UDP-Glc into a transition state that leads to product formation.
ABO Blood-Group System↗
Synthesis of reference standards to enable single cell metabolomic studies of tetramethylrhodamine-labeled ganglioside GM1.
Ganglioside GM1 and its seven potential catabolic products: asialo-GM1, GM2, asialo-GM2, GM3, Lac-Cer, Glc-Cer and Cer, were labeled with tetramethylrhodamine (TMR) to permit ultra-sensitive analysis using laser-induced fluorescence (LIF) detection. The preparation involved acylation of the homogenous C(18)lyso-forms of GM1, Lac-Cer, Glc-Cer and Cer with the N-hydroxysuccinimide ester of a beta-alanine-tethered 6-TMR derivative, followed by conversion of these labeled products using galactosidase, sialidase, and sialyltransferase enzymes. The TMR-glycolipid analogs produced are detectable on TLC down to the 1 ng level by the naked eye. All eight compounds could be separated within 4 min in capillary electrophoresis where they could be detected at the zeptomole (ca. 1000 molecule) level using LIF.
Animals↗
Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis.
The major structural component of the cell wall of Mycobacterium tuberculosis is a lipidated polysaccharide, the mycoyl-arabinogalactan-peptidoglycan (mAGP) complex. This glycoconjugate plays a key role in the survival of the organism, and thus, enzymes involved in its biosynthesis have attracted attention as sites for drug action. At the core of the mAGP is a galactan composed of D-galactofuranose residues attached via alternating beta-(1-->5) and beta-(1-->6) linkages. A single enzyme, glfT, has been shown to synthesize both glycosidic linkages. We report here the first high-level expression and purification of glfT by expression of the Rv3808c gene in Escherichia coli C41(DE3). Following a three-step purification procedure, 3-7 mg of protein of >95% purity was isolated from each liter of culture. We subsequently probed the substrate specificity of glfT by evaluating a panel of potential mono- and oligosaccharide substrates and demonstrated, for the first time, that trisaccharides are better substrates than disaccharides and that one disaccharide, in which the terminal D-galactofuranose residue is replaced with an L-arabinofuranose moiety, is a weak substrate. Kinetic characterization of the enzyme using four of the oligosaccharide acceptors gave K(m) values ranging from 204 microM to 1.7 mM. Through the use of NMR spectroscopy and mass spectrometry, we demonstrated that this recombinant enzyme, like the wild-type protein, is bifunctional and can synthesize both beta-(1-->6) and beta-(1-->5)-linkages in an alternating fashion. Access to purified glfT is expected to facilitate the development of high-throughput assays for the identification of inhibitors of the enzyme, which are potential antituberculosis agents.
Bridged Bicyclo Compounds, Heterocyclic↗
Synthesis of n-octyl 2,6-dideoxy-alpha-L-lyxo-hexopyranosyl-(1-->2)-3-amino-3-deoxy-beta-D-galactopyranoside, an analog of the H-disaccharide antigen.
The synthesis of an analog of the H-disaccharide antigen (2), in which the galactopyranosyl moiety bears an amino group at C-3 and the fucopyranosyl residue is deoxygenated at C-2, is reported. The key reaction in the preparation of 2 was the glycosylation of an appropriately protected n-octyl 3-azido-3-deoxy-galactopyranoside derivative with a 2,6-dideoxy thioglycoside promoted by 1-(phenylsulfinyl)piperidine and triflic anhydride. Disaccharide 2 is of interest in studies directed towards understanding the molecular basis of substrate recognition by the blood group A and B glycosyltransferases.
Antigens, Bacterial↗
Carbohydrate recognition by Clostridium difficile toxin A.
Clostridium difficile TcdA is a large toxin that binds carbohydrates on intestinal epithelial cells. A 2-A resolution cocrystal structure reveals two molecules of alpha-Gal-(1,3)-beta-Gal-(1,4)-beta-GlcNAcO(CH(2))(8)CO(2)CH(3) binding in an extended conformation to TcdA. Residues forming key contacts with the trisaccharides are conserved in all seven putative binding sites in TcdA, suggesting a mode of multivalent binding that may be exploited for the rational design of novel therapeutics.
Bacterial Toxins↗
Purification, kinetic characterization, and mapping of the minimal catalytic domain and the key polar groups of Helicobacter pylori alpha-(1,3/1,4)-fucosyltransferases.
The minimal catalytic domain of alpha-(1,3/1,4)-fucosyltransferases (FucTs) from Helicobacter pylori strains NCTC11639 and UA948 was mapped by N- and C-terminal truncations. Only the C terminus could be truncated without significant loss of activity. 11639FucT and UA948FucT contain 10 and 8 heptad repeats, respectively, which connect the catalytic domain with the C-terminal putative amphipathic alpha-helices. Deletion of all heptad repeats almost completely abolished enzyme activity. Nevertheless, with only one heptad repeat 11639FucT is fully active, whereas UA948FucT is partially active. Removal of the two putative amphipathic alpha-helices dramatically increased protein expression and solubility, enabling purification with yields of milligrams/liter. Steady-state kinetic analysis of the purified FucTs showed that 11639FucTs possessed slightly tighter binding affinity for both Type II acceptor and GDP-fucose donor than UA948FucT, and its kcat of 2.3 s(-1) was double that of UA948FucT, which had a kcat value of 1.1 s(-1) for both Type II and Type I acceptors. UA948FucT strongly favors Type II over the Type I acceptor with a 20-fold difference in acceptor Km. Sixteen modified Type I and Type II series acceptors were employed to map the molecular determinants of acceptors required for recognition by H. pylori alpha-(1,3/1,4)-FucTs. Deoxygenation at 6-C of the galactose in Type II acceptor caused a 5000-fold decrease in alpha1,3 activity, whereas in Type I acceptor this completely abolished alpha1,4 activity, indicating that this hydroxyl group is a key polar group.
Catalytic Domain↗
The cis-AB blood group phenotype: fundamental lessons in glycobiology.
The cis-AB phenotype can raise questions about an apparently paradoxical inheritance of the ABO blood group, such as the birth of an O child from an AB mother. These subtype ABO alleles confer the ability to create both A and B antigens with a single enzyme. A variety of different cis-AB enzymes have been reported and many feature an interchange of amino acids between the normal A enzyme sequence and its B counterpart, rendering the mutant enzyme capable of creating both antigens. The resulting red blood cells do not usually express A or B antigen at the same level that would be expected on common group A(1) or B red blood cells, and the results of investigations into the kinetics of the cis-AB enzyme more clearly predict the extent of antigen expression. By correctly identifying the cis-AB phenotype, the blood bank can be of assistance to a clinician faced with a patient with what appears to be a genetically impossible ABO blood group.
ABO Blood-Group System↗
Computational design and experimental evaluation of glycosyltransferase mutants: engineering of a blood type B galactosyltransferase with enhanced glucosyltransferase activity.
Glycosyltransferases are an enormous and diverse class of enzyme encompassing 1% of all sequenced genomes. They catalyze the transfer of a monosaccharide from an activated donor such as a sugar-nucleotide to an acceptor molecule. Though the primary sequences of glycosyltransferases have little homology, X-ray structural studies on glycosyltransferases have revealed that there are two main folds and that the orientation of the sugar donors with respect to the folds is highly conserved. It seems that glycosyltransferases have evolved diversified specificities toward donor sugars by changing the amino acids around the monosaccharide moiety without altering the orientation of the nucleotide moiety. In this study, we designed new glycosyltransferases with altered donor specificities by use of a novel empirical model called the Epimer Propensity Index (EPI). The EPI was constructed using 221 carbohydrate-protein complex structures in the Protein Data Bank with either galactose or glucose in the complex. The blood type B synthesizing glycosyltransferase GTB, a galactosyltransferase was our target enzyme. Two GTB mutants designed to exhibit enhanced glucosyltransferase activity were cloned, expressed and characterized experimentally. The predicted GTB mutants, Ser185Asn and Ser185Cys, exhibited 4.3- and 4.8-fold elevations in k(cat)/K(m) for UDP-Glc relative to that of wild-type enzyme.
Amino Acid Substitution↗
Differential recognition of the type I and II H antigen acceptors by the human ABO(H) blood group A and B glycosyltransferases.
The human ABO(H) blood group A and B antigens are generated by the homologous glycosyltransferases A (GTA) and B (GTB), which add the monosaccharides GalNAc and Gal, respectively, to the cell-surface H antigens. In the first comprehensive structural study of the recognition by a glycosyltransferase of a panel of substrates corresponding to acceptor fragments, 14 high resolution crystal structures of GTA and GTB have been determined in the presence of oligosaccharides corresponding to different segments of the type I (alpha-l-Fucp-(1-->2)-beta-D-Galp-(1-->3)-beta-D-GlcNAcp-OR, where R is a glycoprotein or glycolipid in natural acceptors) and type II (alpha-l-Fucp-(1-->2)-beta-D-Galp-(1-->4)-beta-d-GlcNAcp-OR) H antigen trisaccharides. GTA and GTB differ in only four "critical" amino acid residues (Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268). As these enzymes both utilize the H antigen acceptors, the four critical residues had been thought to be involved strictly in donor recognition; however, we now report that acceptor binding and subsequent transfer are significantly influenced by two of these residues: Gly/Ser-235 and Leu/Met-266. Furthermore, these structures show that acceptor recognition is dominated by the central Gal residue despite the fact that the L-Fuc residue is required for efficient catalysis and give direct insight into the design of model inhibitors for GTA and GTB.
ABO Blood-Group System↗
High-throughput screening for enzyme inhibitors using frontal affinity chromatography with liquid chromatography and mass spectrometry.
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 Kd value of 0.3 microM. Other binders yielded Kd values ranging from 0.35 to 3.35 microM. 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.
Chromatography, Affinity↗
A single aromatic amino acid at the carboxyl terminus of Helicobacter pylori {alpha}1,3/4 fucosyltransferase determines substrate specificity.
Fucosyltransferases (FucT) from different Helicobacter pylori strains display distinct Type I (Galbeta1,3GlcNAc) or Type II (Galbeta1,4GlcNAc) substrate specificity. FucT from strain UA948 can transfer fucose to the OH-3 of Type II acceptors as well as to the OH-4 of Type I acceptors on the GlcNAc moiety, so it has both alpha1,3 and alpha1,4 activities. In contrast, FucT from strain NCTC11639 has exclusive alpha1,3 activity. Our domain swapping study (Ma, B., Wang, G., Palcic, M. M., Hazes, B., and Taylor, D. E. (2003) J. Biol. Chem. 278, 21893-21900) demonstrated that exchange of the hypervariable loops, (347)DNPFIFC(353) in 11639FucT and (345)CNDAHYSALH(354) in UA948FucT, were sufficient to either confer or abolish alpha1,4 activity. Here we performed alanine scanning site-directed mutagenesis to identify which amino acids within (345)CNDAHYSALH(354) of UA948FucT confer Type I substrate specificity. The Tyr(350) --> Ala mutation dramatically reduced alpha1,4 activity without lowering alpha1,3 activity. None of the other alanine substitutions selectively eliminated alpha1,4 activity. To elucidate how Tyr(350) determines alpha1,4 specificity, mutants Tyr(350) --> Phe, Tyr(350) --> Trp, and Tyr(350) --> Gly were constructed in UA948FucT. These mutations did not decrease alpha1,3 activity but reduced the alpha1,4 activity to 66.9, 55.6, and 3.1% [corrected] of wild type level, respectively. Apparently the aromatic nature, but not the hydroxyl group of Tyr(350), is essential for alpha1,4 activity. Our data demonstrate that a single amino acid (Tyr(350)) in the C-terminal hypervariable region of UA948FucT determines Type I acceptor specificity. Notably, a single aromatic residue (Trp) has also been implicated in controlling Type I acceptor preference for human FucT III, but it is located in an N-terminal hypervariable stem domain.
Amino Acid Sequence↗
Multiple sampling in single-cell enzyme assays using CE-laser-induced fluorescence to monitor reaction progress.
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 microm 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, alpha1,3-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 alpha-glucosidase I activity.
Animals↗
The importance of disordered loops in ABO glycosyltransferases.
The human ABO antigens are carbohydrates that differ from each other by the immunodominant sugar. The O phenotype is characterized by the absence of the A- or B-defining carbohydrate. The glycosyltransferases that create the A and B antigens share a considerable amino acid sequence and a structural homology and feature 2 series of amino acids whose exact location within the enzymes' structure cannot be determined. One series is 16 amino acids in length and probably lies next to the catalytic center, whereas less is known about the other 10-amino acid disordered loop located at the C-terminus of the protein. These "disordered" segments of amino acids can be found in other glycosyltransferases from disparate species. The precise role of these amino acids is unclear although recent evidence suggests that they are involved in substrate binding and turnover. A more complete understanding of its function will provide fundamental insights into the activity of glycosyltransferases and a potential target for novel therapeutics in the case of pathogens. In this review, we describe the nature of various disordered regions in glycosyltransferase structures from bacteria to human beings.
ABO Blood-Group System↗
Amino-acid substitution in the disordered loop of blood group B-glycosyltransferase enzyme causes weak B phenotype.
BACKGROUND: Few studies have investigated the reaction kinetics and interactions with nucleotide donor and acceptor substrates of mutant human ABO glycosyltransferases. Previous work identified a B(w) allele featuring a 556G>A polymorphism giving rise to a weak B phenotype. This polymorphism is predicted to cause a M186V amino-acid mutation within a highly conserved series of 16 amino acids present both in both blood group A- and blood group B-synthesizing enzymes. These residues are known as the disordered loop because their location cannot be determined in the crystal structure of the enzyme. Another patient has been identified with a 556G>A B(w) allele and the kinetics of the resulting mutant glycosyltransferase were studied. STUDY DESIGN AND METHODS: Serologic testing with murine and human reagents, amplification of the coding regions of exons 6 and 7, and DNA sequencing were performed with standard protocols. Enzyme kinetic studies utilized a model of human GTB M186V expressed in Escherichia coli with radiolabeled UDP-galactose and UDP-N-acetylgalactosamine as donor substrates and synthetic H-disaccharide as acceptor following standard protocols. RESULTS: The patient's red blood cells demonstrated a weak, but not mixed-field, B phenotype. Kinetic studies on the mutant enzyme revealed diminished activity (k(cat) = 0.15 per sec with UDP-galactose compared to 5.1 per sec for wild-type GTB) and elevated K(m) values for all substrates. CONCLUSION: This enzyme with a mutation in the disordered loop produces weak B antigen expression because of greatly decreased enzyme activity and reduced affinity for B-donor and acceptor substances.
ABO Blood-Group System↗
Structural basis for the inactivity of human blood group O2 glycosyltransferase.
The human ABO(H) blood group antigens are carbohydrate structures generated by glycosyltransferase enzymes. Glycosyltransferase A (GTA) uses UDP-GalNAc as a donor to transfer a monosaccharide residue to Fuc alpha1-2Gal beta-R (H)-terminating acceptors. Similarly, glycosyltransferase B (GTB) catalyzes the transfer of a monosaccharide residue from UDP-Gal to the same acceptors. These are highly homologous enzymes differing in only four of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. Blood group O usually stems from the expression of truncated inactive forms of GTA or GTB. Recently, an O(2) enzyme was discovered that was a full-length form of GTA with three mutations, P74S, R176G, and G268R. We showed previously that the R176G mutation increased catalytic activity with minor effects on substrate binding. Enzyme kinetics and high resolution structural studies of mutant enzymes based on the O(2) blood group transferase reveal that whereas the P74S mutation in the stem region of the protein does not appear to play a role in enzyme inactivation, the G268R mutation completely blocks the donor GalNAc-binding site leaving the acceptor binding site unaffected.
ABO Blood-Group System↗
Large-scale chemical and chemo-enzymatic synthesis of a spacer-containing Pk-trisaccharide.
The Pk-trisaccharide, linked to a solid carrier, is a potential agent for neutralization of shiga-like toxin in the gastrointestinal tract. Two approaches to the multigram-scale synthesis of a linkable Pk-trisaccharide derivative were therefore investigated. A four-step chemical synthesis yielded 8-methoxycarbonyloctyl beta-lactoside in 75% yield from lactose. Further conversion of this derivative through either multistep organic synthesis or one-step enzymatic galactosylation with UDP-galactose and recombinant alpha-1,4-galactosyltransferase gave the Pk-trisaccharide derivative 8-methoxycarbonyloctyl alpha-D-galactopyranosyl-(1-->4)-beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranoside in 25% and 68% overall yields from commercial lactose, respectively.
Acetic Acid↗
Glycosyltransferase-catalyzed synthesis of thiooligosaccharides.
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Carbohydrate Conformation↗