Naphthyl Thio- and Carba-xylopyranosides for Exploration of the Active Site of ß-1,4-Galactosyltransferase 7 (ß4GalT7).

Xyloside analogues with substitution of the endocyclic oxygen atom by sulfur or carbon were investigated as substrates for ß-1,4-galactosyltransferase 7 (ß4GalT7), a key enzyme in the biosynthesis of glycosaminoglycan chains. The analogues with an endocyclic sulfur atom proved to be excellent substrates for ß4GalT7, and were galactosylated approximately fifteen times more efficiently than the corresponding xyloside. The 5a-carba-ß-xylopyranoside in the d-configuration proved to be a good substrate for ß4GalT7, whereas the enantiomer in the l-configuration showed no activity. Further investigations by X-ray crystallography, NMR spectroscopy, and molecular modeling provided a rationale for the pronounced activity of the sulfur analogues. Favorable π-π interactions between the 2-naphthyl moiety and a tyrosine side chain of the enzyme were observed for the thio analogues, which open up for the design of efficient GAG primers and inhibitors.

Crystal structure of the catalytic domain of Drosophila beta1,4-Galactosyltransferase-7.

The beta1,4-galactosyltransferase-7 (beta4Gal-T7) enzyme, one of seven members of the beta4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member beta4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of beta4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 A resolution. In the crystal structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of beta4Gal-T7 and beta4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH(2)OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the beta4Gal-T7 crystal structure shows that the aromatic side chain of Tyr(177) interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.

Highly oriented recombinant glycosyltransferases: site-specific immobilization of unstable membrane proteins by using Staphylococcus aureus sortase A.

Recombinant glycosyltransferases are potential biocatalysts for the construction of a compound library of oligosaccharides, glycosphingolipids, glycopeptides, and various artificial glycoconjugates on the basis of combined chemical and enzymatic synthetic procedures. The structurally defined glycan-related compound library is a key resource both in the basic studies of their functional roles in various biological processes and in the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that the immobilization of extremely unstable membrane-bound glycosyltransferases on some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile separation of products and recycling use of enzymes possible. Until now, however, it seems that no standardized protocol preventing a significant loss of enzyme activity is available due to the lack of a general method of site-selective anchoring between glycosyltransferases and scaffold materials through a stable covalent bond. Here we communicate a versatile and efficient method for the immobilization of recombinant glycosyltransferases onto commercially available solid supports by means of transpeptidase reaction by Staphylococcus aureus sortase A. This protocol allowed for the first time highly specific conjugation at the designated C-terminal signal peptide moiety of recombinant human beta1,4-galactosyltransferase or recombinant Helicobacter pylori alpha1,3-fucosyltransferase with simple aliphatic amino groups displayed on the surface of solid materials. Site-specifically immobilized enzymes exhibited the desired sugar transfer activity, an improved stability, and a practical reusability required for rapid and large-scale synthesis of glycoconjugates. Considering that most mammalian enzymes responsible for the posttranslational modifications, including the protein kinase family, as well as glycosyltransferases are unstable and highly oriented membrane proteins, the merit of our strategy based on "site-specific" transpeptidation is evident because the reaction proceeds only at an engineered C-terminus without any conformational influence around the active sites of both enzymes as well as heptad repeats of rHFucT required to maintain native secondary and quaternary structures during the dimerization on cell surfaces.

Synthesis and photolytic activation of 6''-O-2-nitrobenzyl uridine-5'-diphosphogalactose: a 'caged' UDP-Gal derivative.

Placing an 2-nitrobenzyl group on O-6 of the galactosyl residue in uridine-5'-diphosphogalactose (UDP-Gal) gives 6''-O-2-nitrobenzyl-UDP-Gal that is shown to be inactive as a donor substrate for beta-(1-->4)-galactosyltransferase (GalT). On irradiation at 365 nm, the nitrobenzyl group is completely removed yielding native UDP-Gal that then transfers normally to produce the expected betaGal-(1-->4)-betaGlcNAc disaccharidic linkage. 6''-O-2-Nitrobenzyl-UDP-Gal thus fulfils the minimum requirements of a 'caged' UDP-Gal for application in time-resolved crystallographic studies of beta-(1-->4)-GalT.

Structural snapshots of beta-1,4-galactosyltransferase-I along the kinetic pathway.

During the catalytic cycle of beta1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn(2+) followed by UDP-Gal, two flexible loops, a long and a short loop, change their conformation from open to closed. We have determined the crystal structures of a human M340H-Gal-T1 mutant in the open conformation (apo-enzyme), its Mn(2+) and Mn(2+)-UDP-Gal-bound complexes, and of a pentenary complex of bovine Gal-T1-Mn(2+)-UDP-GalNAc-Glc-alpha-lactalbumin. These studies show that during the conformational changes in Gal-T1, the coordination of Mn(2+) undergoes significant changes. It loses a coordination bond with a water molecule bound in the open conformation of Gal-T1 while forming a new coordination bond with another water molecule in the closed conformation, creating an active ground-state structure that facilitates enzyme catalysis. In the crystal structure of the pentenary complex, the N-acetylglucosamine (GlcNAc) moiety is found cleaved from UDP-GalNAc and is placed 2.7A away from the O4 oxygen atom of the acceptor Glc molecule, yet to form the product. The anomeric C1 atom of the cleaved GalNAc moiety has only two covalent bonds with its non-hydrogen atoms (O5 and C2 atoms), similar to either an oxocarbenium ion or N-acetylgalactal form, which are crystallographically indistinguishable at the present resolution. The structure also shows that the newly formed, metal-coordinating water molecule forms a hydrogen bond with the beta-phosphate group of the cleaved UDP moiety. This hydrogen bond formation results in the rotation of the beta-phosphate group of UDP away from the cleaved GalNAc moiety, thereby preventing the re-formation of the UDP-sugar during catalysis. Therefore, this water molecule plays an important role during catalysis in ensuring that the catalytic reaction proceeds in a forward direction.

Structure and catalytic cycle of beta-1,4-galactosyltransferase.

Beta-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle.

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.

Triggering loops and enzyme function: identification of loops that trigger and modulate movements.

Enzyme function often involves a conformational change. There is a general agreement that loops play a vital role in correctly positioning the catalytically important residues. Nevertheless, predicting the functional loops and most importantly their role in enzyme function remains a difficult task. A major reason for this difficulty is that loops that undergo conformational change are frequently not well conserved in their primary sequence. beta1,4-Galactosyltransferase is one such enzyme. There, the amino acid sequence of a long loop that undergoes a large conformational change upon substrate binding is not well conserved. Our molecular dynamics simulations show that the large conformational change in the long loop is brought about by a second, interacting loop. Interestingly, while the structural change of the second loop is much smaller than that of the long loop, its sequence (particularly glycine residues) is highly conserved. We further examine the generality of the proposition that there are loops that trigger movements but nevertheless show little or no structural changes in crystals. We focus on two other enzymes, enolase and lipase. We chose these enzymes, since they too undergo conformational change upon ligand binding, however, they have different folds and different functions. Through multiple sets of simulations we show that the conformational change of the functional loop(s) is brought about through communication of flexibility by triggering loops that have several glycine residues. We further propose that similar to the conservation of common favorable fold types and structural motifs, evolution has also conserved common "skillful" mechanisms. Mechanisms may be conserved across different folds, sequences and functions, with adaptation to specific enzymatic roles.

Quantitative electrospray mass spectrometry for the rapid assay of enzyme inhibitors.

BACKGROUND: Combinatorial chemistry has become an important method for identifying effective ligand-receptor binding, new catalysts and enzyme inhibitors. In order to distinguish the most active component of a library or to obtain structure-activity relationships of compounds in a library, an efficient quantitative assay is crucial. Electrospray mass spectrometry has become an indispensable tool for qualitatively screening combinatorial libraries and its use for quantitative analysis has recently been demonstrated. RESULTS: This paper describes the use of quantitative electrospray mass spectrometry for screening libraries of inhibitors of enzymatic reactions, specifically the enzymatic glycosylation by beta-1,4-galactosyltransferase, which catalyzes the transfer of galactose from uridine-5'-diphosphogalactose to the 4-position of N-acetylglucosamine beta OBn (Bn: benzene) to form N-acetyllactosamine beta OBn. Our mass spectrometric screening approach showed that both nucleoside diphosphates and triphosphates inhibited galactosyltransferase while none of the nucleoside monophosphates, including uridine-5'-monophosphate, showed any inhibition. Additional libraries were generated in which the concentrations of the inhibitors were varied and, using mass spectrometry, uridine-5'-diphosphate-2-deoxy-2-fluorogalactose was identified as the best inhibitor. CONCLUSIONS: This report introduces quantitative electrospray mass spectrometry as a rapid, sensitive and accurate quantitative assaying tool for inhibitor libraries that does not require a chromophore or radiolabeling. A viable alternative to existing analytical techniques is thus provided. The new technique will greatly facilitate the discovery of novel inhibitors against galactosyltransferase, an enzyme for which there are few potent inhibitors.

Analysis of the sequences of human beta-1,4-galactosyltransferase cDNA clones.

UDP-galactose:beta-1,4 N-acetyl glucosamine galactosyltransferase (4 beta GT) is a promising tumor marker for ovarian cancer. To study the role of 4 beta GT in malignant transformation at the molecular level human 4 beta GT cDNA and genomic clones were isolated and analyzed. For the isolation of 4 beta GT cDNA and genomic clones, a human fetal liver cDNA library in lambda gt11 and a human genomic library in EMBL-3B vectors respectively were screened using a 4 beta GT cDNA insert as the probe. Complete sequence of the cDNA clones were determined by subcloning in plasmid vectors, and compared with the published sequence of human liver 4 beta GT. Presence of various 4 beta GT exons in the genomic clones were determined by Southern blot analysis using specific oligodeoxynucleotide probes. Among the 5 cDNA clones isolated, 2 clones GTN 6 and GTN 17 were sibling clones and had a nucleotide sequence identical to the published 4 beta GT cDNA sequence, except at the 3'-end, where these clones had 7 unique nucleotide sequences. One cDNA clone, GTN2 also had a nucleotide sequence identical to that of 4 beta GT, except for 3 G residues at the 5'-end. One cDNA clone, GTN 1, had a unique sequence at the 5'-end comprising of 74 nucleotides. Another clone, GTN 20, was unrelated to 4 beta GT. Analysis of genomic clones showed that 4 beta GT exons 3, 4, 5 and 6 were present in a 14 kb genomic clone, EMGT-4. Exon 1 was present in a separate 16 kb clone, EMGT-6.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Highly efficient oligosaccharide synthesis on water-soluble polymeric primers by recombinant glycosyltransferases immobilised on solid supports.

Recombinant beta-1,4-galactosyltranferase (beta 1,4-GalT) and alpha-2,6-sialytransferase (alpha 2,6-SiaT) immobilised covalently with activated Sepharose beads were employed for the practical synthesis of a trisaccharide derivative, Neu-5Ac alpha(2-->6)Gal beta(1-->4)GlcNAc beta-O-(CH2)6-NH2, on a water-soluble primer having GlcNAc residues through a alpha-chymotrypsin-sensitive linker.

Further evidence for an active site polypeptide of galactosyl transferase.

In a previous report it was shown that galactosyl transferase activity after blotting from acrylamide gel was present in a molecular weight range of less than 14 kDa, in Triton X-100 (1). Molecular sieve chromatography on Superose 12, in the presence of Triton X-100, gave the same result. The low molecular weight activity peak was eluted together with peptides as a part of the covalent structure of the enzyme or as absolutely requires effectors. Peptide mapping showed a new poly-lysine-like peptide and a new hydrophobic peptide in this low molecular weight activity peak as effectors of the enzyme inside its hydrophobic environment.

UDP-6-deoxy-6-fluoro-alpha-D-galactose binds to two different galactosyltransferases, but neither can effectively catalyze transfer of the modified galactose to the appropriate acceptor.

The effect of substitution of the HO-6 of D-galactose with fluorine on the ability of alpha-(1-->3)-galactosyltransferase (EC 2.4.1.151) and beta-(1-->4)-galactosyltransferase (EC 2.4.1.22) to catalyze its transfer from UDP to an appropriate acceptor was determined. HPLC analyses indicated that each transferase properly catalyzed formation of the expected product [beta-D-Gal-(1-->4)-D-GlcNAc] for the beta-(1-->4)-galactosyltransferase and alpha-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-D-GlcNAc for the alpha-(1-->3)-D-galactosyltransferase] when UDP-alpha-D-Gal was the substrate. When UDP-6-deoxy-6-fluoro-alpha-D-galactose (6) was used in conjunction with each transferase, no product indicative of transfer of 6-deoxy-6-fluoro-D-galactose to its respective acceptor sugar was identified. 6-Deoxy-6-fluoro-D-galactose (3) was obtained by hydrolysis of methyl 6-deoxy-6-fluoro-alpha-D-galactopyranoside, synthesized by the selective fluorination of methyl alpha-D-galactopyranoside with diethylaminosulfur trifluoride (DAST), with aqueous trifluoroacetic acid. Acetylation of 3 gave crystalline 1,2,3,4-tetra-O-acetyl-6-deoxy-6-fluoro-beta-D-galactopyranose, which was converted to the corresponding 1-alpha-phosphate and used for the synthesis of 6.

Galactosyl transfer ability of beta-(1-->4)-galactosyltransferase toward 5a-carba-sugars.

Bovine beta-(1-->4)-galactosyltransferase was assayed with a series of 5a-carba-sugars, i.e., sugar analogues in which the ring oxygen of pyranose is replaced by a methylene group. The analogues are 5a-carba-sugar of 2-acetamido-2-deoxy-alpha-DL-galactopyranose, both alpha and beta anomers of 2-acetamido-2-deoxy-DL-glucopyranose (5a-carba-DL-GlcNAc), and 2-acetamido-2-deoxy-DL-mannopyranose. Of these analogues, both alpha and beta anomers of 5a-carba-DL-GlcNAc act as an acceptor. Enzymatic synthesis using the alpha and beta anomers of 5a-carba-DL-GlcNAc afforded the corresponding D-Gal-beta-(1-->4)-5a-carba-alpha-D-GlcNAc and D-Gal-beta-(1-->4)-5a-carba-beta-D-GlcNAc on a practical scale, and these structures were confirmed by NMR spectroscopy. These results indicate that the ring oxygen atom in the 5a-carba-D-GlcNAc is not used for specific recognition by bovine beta-(1-->4)-galactosyltransferase.

Cloning and GST-fused expression in E. coli of mouse beta-1,4-galactosyltransferase.

Beta-1,4-galactosyltransferase (beta4Gal-T) (EC 2.4.1.38) plays a multifunctional role in many aspects of normal cell physiology. By now, several dozens of beta4Gal-T genes have been cloned, separated from mouse, chick, bovine, human, etc. This paper presents the cloning and GST-fused expression of mouse beta4Gal-T gene in Escherichia coli (E. coli). The target gene was cloned by PCR, followed by identification by DNA sequencing and expression in E.coli with isopropyl-beta-D-thiogalactoside (IPTG) gradient concentrations, products of which were separated on SDS-PAGE showing that the target protein had the same molecular weight as that of mouse beta4Gal-T. The transcriptional product of beta4Gal-T gene was proved by Western hybridization analysis to be due to GST-fusion.

A microscopic insight from conformational thermodynamics to functional ligand binding in proteins.

We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for ß-1,4-galactosyltransferase (ß4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock ß4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.

In vitro folding of ß-1,4galactosyltransferase and polypeptide-α-N-acetylgalactosaminyltransferase from the inclusion bodies.

The aim of this article is to present a unique in vitro folding technique for glycosyltransferases to generate active proteins that can be used for X-ray crystallographic and bioconjugation protocols. Although a number of in vitro refolding methods are available, ß1,4galactosyltransferases in large quantities can be made using the current protocol only. This technique is not only limited to glycosyltransferases alone but has been successfully used to refold single-chain antibodies and other molecules. Although this in vitro folding method is quite similar to other methods, it differs from them by the use of S-sulfonation of the inclusion bodies before setting up the in vitro refolding of the protein.

Ionic-liquid-based catch and release mass spectroscopy tags for enzyme monitoring.

A novel, inexpensive and versatile ionic-liquid-based catch and release mass spectrometry tag (I-Tag) that facilitates substrate purification, fast, robust and sensitive enzymatic reaction monitoring and quantitative kinetic analysis has been developed. The applicability of the system has been demonstrated in an enzymatic assay with ß-1,4-galactosyltransferase.