Soluble Glycosyltransferases Research Articles

Enzymatic Glyco-Modification of Synthetic Membrane Systems.

The present report assesses the capability of a soluble glycosyltransferase to modify glycolipids organized in two synthetic membrane systems that are attractive models to mimic cell membranes: giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs). The objective was to synthesize the Gb3 antigen (Galα1,4Galβ1,4Glcβ-Cer), a cancer biomarker, at the surface of these membrane models. A soluble form of LgtC that adds a galactose residue from UDP-Gal to lactose-containing acceptors was selected. Although less efficient than with lactose, the ability of LgtC to utilize lactosyl-ceramide as an acceptor was demonstrated on GUVs and SLBs. The reaction was monitored using the B-subunit of Shiga toxin as Gb3-binding lectin. Quartz crystal microbalance with dissipation analysis showed that transient binding of LgtC at the membrane surface was sufficient for a productive conversion of LacCer to Gb3. Molecular dynamics simulations provided structural elements to help rationalize experimental data.

Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803

Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66mg/L of p-hydroxyphenylacetaldoxime and 5mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation.

核移行糖転移酵素を用いた核内<i>N</i>-アセチルグルコサミン修飾タンパク質同定法の確立

核移行糖転移酵素を用いた核内<i>N</i>-アセチルグルコサミン修飾タンパク質同定法の確立

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Pseudomonas aeruginosa produces three exopolysaccharides, Psl, Pel, and alginate, that play vital roles in biofilm formation. Pel is a glucose-rich, cellulose-like exopolysaccharide. The essential Pel biosynthesis proteins are encoded by seven genes, pelA to pelG. Bioinformatics analysis suggests that PelF is a cytosolic glycosyltransferase. Here, experimental evidence was provided to support this PelF function. A UDP-glucose dehydrogenase-based assay was developed to quantify UDP-glucose. UDP-glucose was proposed as the substrate for PelF. The isogenic pelF deletion mutant accumulated 1.8 times more UDP-glucose in its cytosol than the wild type. This suggested that PelF, which was found localized in the cystosol, uses UDP-glucose as substrate. Additionally, in vitro experiments confirmed that PelF uses UDP-glucose as substrate. To analyze the functional roles of conserved residues in PelF, site-directed mutagenesis was performed. The presence of the EX7E motif is characteristic for various glycosyltransferase families, and in PelF, E405/E413 are the conserved residues in this motif. Replacement of E405 with A resulted in a reduction of PelF activity to 30.35% ± 3.15% (mean ± standard deviation) of the wild-type level, whereas replacement of the second E, E413, with A did not produce a significant change in the activity of PelF. Moreover, replacement of both E residues did not result in a loss of PelF function, but replacement of the conserved R325 or K330 with A resulted in a complete loss of PelF activity. Overall, our data show that PelF is a soluble glycosyltransferase that uses UDP-glucose as the substrate for Pel synthesis and that conserved residues R325 and K330 are important for the activity of PelF.

The origin and function of platelet glycosyltransferases

AbstractPlatelets are megakaryocyte subfragments that participate in hemostatic and host defense reactions and deliver pro- and antiangiogenic factors throughout the vascular system. Although they are anucleated cells that lack a complex secretory apparatus with distinct Golgi/endoplasmic reticulum compartments, past studies have shown that platelets have glycosyltransferase activities. In the present study, we show that members of 3 distinct glycosyltransferase families are found within and on the surface of platelets. Immunocytology and flow cytometry results indicated that megakaryocytes package these Golgi-derived glycosyltransferases into vesicles that are sent via proplatelets to nascent platelets, where they accumulate. These glycosyltransferases are active, and intact platelets glycosylate large exogenous substrates. Furthermore, we show that activation of platelets results in the release of soluble glycosyltransferase activities and that platelets contain sufficient levels of sugar nucleotides for detection of glycosylation of exogenously added substrates. Therefore, the results of the present study show that blood platelets are a rich source of both glycosyltransferases and donor sugar substrates that can be released to function in the extracellular space. This platelet-glycosylation machinery offers a pathway to a simple glycoengineering strategy improving storage of platelets and may serve hitherto unknown biologic functions.

β-Galactoside α2,6-Sialyltransferase I Cleavage by BACE1 Enhances the Sialylation of Soluble Glycoproteins

BACE1 (beta-site amyloid precursor protein-cleaving enzyme-1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, amyloid beta-peptide, and has been implicated in triggering the pathogenesis of Alzheimer disease. We showed previously that BACE1 cleaves beta-galactoside alpha2,6-sialyltransferase I (ST6Gal I) to initiate its secretion, but it remained unclear how BACE1 affects the cellular level of alpha2,6-sialylation. Here, we found that BACE1 overexpression in Hep3B cells increased the sialylation of soluble secreted glycoproteins, but did not affect cell-surface sialylation. The sialylation of soluble glycoproteins was not increased by ST6Gal I overexpression alone, but was increased by co-overexpression of ST6Gal I and BACE1 or by expression of the soluble form of ST6Gal I, suggesting that soluble ST6Gal I produced by BACE1 plays, at least in part, a role in the sialylation of soluble glycoproteins. We also found that plasma glycoproteins from BACE1-deficient mice exhibited reduced levels of alpha2,6-sialylation compared with those from wild-type mice. We propose a novel regulatory mechanism in which cleavage and secretion of ST6Gal I enhance the sialylation of soluble glycoprotein substrates.

Enzymes Responsible for Synthesis of Corneal Keratan Sulfate Glycosaminoglycans

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, beta1,3-N-acetylglucosaminyltansferase-7 (beta3GnT7) and beta1,4-galactosyltransferase-4 (beta4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of beta3GnT7/beta4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of beta3GnT7 or beta4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.

High Cationic Charge and Bilayer Interface-Binding Helices in a Regulatory Lipid Glycosyltransferase,

In the prokaryote Acholeplasma laidlawii, membrane bilayer properties are sensed and regulated by two interface glycosyltransferases (GTs), synthesizing major nonbilayer- (alMGS GT) and bilayer-prone glucolipids. These enzymes are of similar structure, as many soluble GTs, but are sensitive to lipid charge and curvature stress properties. Multivariate and bioinformatic sequence analyses show that such interface enzymes, in relation to soluble ones of similar fold, are characterized by high cationic charge, certain distances between small and cationic amino acids, and by amphipathic helices. Varying surface contents of Lys/Arg pairs and Trp indicate different membrane-binding subclasses. A predicted potential (cationic) binding helix from alMGS was structurally verified by solution NMR and CD. The helix conformation was induced by a zwitterionic as well as anionic lipid environment, and the peptide was confined to the bilayer interface. Bilayer affinity of the peptide, analyzed by surface plasmon resonance, was higher than that for soluble membrane-seeking proteins/peptides and rose with anionic lipid content. Interface intercalation was supported by phase equilibria in membrane lipid mixtures, analyzed by 31P NMR and DSC. An analogous, potentially binding helix has a similar location in the structurally determined Escherichia coli cell wall precursor GT MurG. These two helices have little sequence conservation in alMGS and MurG homologues but maintain their amphipathic character. The evolutionary modification of the alMGS binding helix and its location close to the acceptor substrate site imply a functional importance in enzyme catalysis, potentially providing a mechanism by which glycolipid synthesis will be sensitive to membrane surface charge and intrinsic curvature strain.

A simple method to express soluble, highly active cyclodextrin glycosyltransferase in recombinant E . coli

The activity of soluble cyclodextrin glycosyltransferase was increased by 40% without formation of inclusion bodies by induction at low temperature with the addition of 10 mM CaCl2 into the medium, when the corresponding gene was expressed in two recombinant E. coli. Furthermore, the protein expressed at 25 °C had approximately 30% higher specific activity than that expressed at 30 °C. The two different hosts, promoters, and media showed the same result, indicating that this simple method can be used in expressing other proteins.

Two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts.

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3-galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.

A Soluble Form of α1,3-Galactosyltransferase Functions within Cells to Galactosylate Glycoproteins

It has been assumed that membrane-bound glycosyltransferases function within the Golgi apparatus to glycosylate glycoproteins. We now report, however, that a truncated, soluble recombinant form of the murine alpha1,3-galactosyltransferase expressed in human 293 cells is highly efficient and comparable to the full-length enzyme in alpha-galactosylating both newly synthesized membrane-associated and secreted glycoproteins. Although the soluble enzyme was secreted by cells as expected, we also found that the full-length, membrane-associated form was secreted. Unexpectedly, both secreted forms are cleaved identically at two primary sites within the stem region by endogenous protease(s) at the indicated positions in the sequence 73KDWW (downward arrow) FPS (downward arrow) WFKNG. These results demonstrate that the soluble alpha1,3-galactosyltransferase is functional within the cell and that specific proteolysis occurs in the stem region. The widespread occurrence of different soluble glycosyltransferases secreted by cells suggests that normal glycoconjugate biosynthesis may involve cooperation between membrane-bound and soluble enzymes.

Glucosylation and galactosylation of diosgenin and solasodine by soluble glycosyltransferase(s) from Solanum melongena leaves

When the crude, lipid-depleted cytosolic fraction (105 000 g supernatant) from Solanum melongena leaves is incubated with UDP- [ 14C]glucose and diosgenin (a steroidal sapogenin of spirostane type) or solasodine (a steroidal alkaloid of spirosolane type), the main reaction product consists of diosgenin or solasodine monoglucoside. However, a small amount (usually less than 5%) of the corresponding monogalactoside is also synthesized. With UDP- [ 14C]galactose as sugar source, almost equal amounts of monoglucoside and monogalactoside derivatives are formed. These results can be explained by the presence of highly active UDP-glucose 4-epimerase in the investigated enzyme preparation. It was found that this epimerase can be almost completely blocked by an addition of 0.1 mM UDP-xylose to the reaction mixture. The use of this inhibitor allowed comparison of the specificities of glucosylation and galactosylation for some potential steroid acceptors. It was shown that the specificity patterns of both these reactions are very similar, which suggests that a single enzyme catalyses sugar transfer from both UDPGlc and UDPGal. Good substrates for both the glucosyltransferase and galactosyltransferase reactions consist of, apart from diosgenin and solasodine, some other spirostane-type sapogenins structurally related to diosgenin, i.e. tigogenin, yamogenin and hecogenin, as well as tomatidine which is a spirosolane-type steroidal alkaloid closely related to solasodine. In contrast to spirosolane alkaloids, steroidal alkaloids of the solanidane type, i.e. solanidine or demmissidine, are very poor substrates. Sterols are neither glucosylated nor galactosylated by the cytosolic enzyme(s) from S. melongena.

A short chemo-enzymic route to glycosphingolipids using soluble glycosyl transferases

Two commercially available soluble glycosyl transferases, a β-1,4-galactosyl transferase from bovine milk and an α-2,6-sialyl transferase from rat liver, were used for the synthesis of several glycosphingolipids. The synthetic strategy involves the chemical synthesis of 2-azido- and 2-amino-sphingosine glycosides (5, 6, 10 and 11) and further glycosylation of their sugar headgroups to complex glycolipids. The azido- or amino-sphingosine lipid sidechain can then be further elaborated by reduction and/or acylation.

Presence of glycosyltransferase inhibitors in the sera of patients with long-term surviving ABO incompatible (A2 to O) kidney grafts.

In the sera of blood group O recipients with long-term surviving blood group A2 kidney grafts, inhibitors of A-glycosyltransferase (alpha-3-N-acetyl-D-galactosaminyl transferase) have been identified in four out of six patients. The inhibitors seem to be of IgG antibody nature and do not appear until at least 1 year after transplantation. The inhibitors are also active against B-transferase (alpha-3-D-galactosyl transferase) as well as against A-glycosyltransferase of pig origin. The importance of these A-glycosyltransferase inhibitors is not clear. They may have a modifying effect on the antigenic expression of the graft. The immunogenicity of soluble glycosyltransferases is well known and the development of antibodies against them might prevent their enzymatic activity.

Glycosylation of proteins from sugar nucleotides by whole cells. Effect of ammonium chloride treatment on mouse thymocytes.

When thymocytes are treated with iso-osmotic NH4Cl, the sugar incorporation into endogenous acceptors from labelled sugar nucleotides is largely increased compared with that in control thymocytes. This effect was obtained with labelled GDP-mannose, UDP-galactose and CMP-N-acetylneuraminic acid. The stimulation observed with NH4Cl-treated thymocytes does not involve the glycosylation of exogenous acceptors, and it was proved that the NH4Cl treatment (1) does not stimulate glycosyltransferase activities themselves, (2) does not lead to the release of soluble glycosyltransferases as the result of an extensive lysis of the thymocytes and (3) does not cause the emergence of glycosyltransferases at the cell surface. In fact, electron-microscopy observations showed that, although marked changes had occurred in the cytoplasm, the plasma membrane is sufficiently maintained to allow the cell to keep roughly its original shape and to retain the intracellular vesicles. We thus demonstrate that this stimulation is due to an enhancement of the entry of sugar nucleotides into the cell. As demonstrated by the inclusion of Trypan Blue within the cells, and the non-stimulation of glycosylation of exogenous large-molecular-mass acceptors, the effect of NH4Cl seems to be limited to the penetration of small-molecular-sized compounds through the plasma membrane. Thus NH4Cl treatment allows the labelled sugar nucleotides to penetrate the cell and to behave as the cellular pool to be utilized for glycosylation by intracellular vesicles.

The effect of linoleic acid and benzyl alcohol on the activity of glycosyltransferases of rat liver golgi membranes and some soluble glycosyltransferases

The effects of the membrane perturbing reagents linoleic acid and benzyl alcohol on the activities of four rat liver Golgi membrane enzymes, N-acetylglucosaminyl-, N-acetylgalactosaminyl-, galactosyl-, and sialytransferases and several soluble glycosyltransferases, bovine milk galactosyl- and N-acetylglucosaminyltransferases and porcine submaxillary N-acetylgalactosaminyltransferases have been studied. In rat liver Golgi membranes, linoleic acid inhibited the activities of N-acetylgalactosaminyl- and galactosyltransferases by 50% or greater, sialyltransferase by 10–15%, and N-acetylglucosaminyltransferase not at all. The isolated bovine milk N-acetylglucosaminyltransferase and porcine submaxillary N-acetylgalactosylaminyltranferase were not inhibited but bovine milk galactosyltransferase was inhibited by 95% or greater. The inhibition by linoleic acid on Golgi membrane galactosyltransferase appears to be a direct effect of the reagent on the enzyme. Incorporation of bovine milk galactosyltransferase into liposomes formed from saturated phospholipids, DMPC, DPPC, and DSPC (dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine) prevented inhibition of the enzyme activity suggesting that the lipid formed a barrier which did not allow linoleic acid access to the enzyme. The water soluble benzyl alcohol was more effective in inhibiting enzymes of the isolated rat liver Golgi complex. All four glycosyltransferases were inhibited, the N-acetylglucosaminyl- and N-acetylgalactosaminyltransferases by more than 95%. A higher concentration of benzyl alcohol was necessary to inhibit the galactosyltransferases than was required for the other Golgi enzymes. Benzyl alcohol also inhibited the isolated bovine milk N-acetylglucosaminyl- and galactosyltransferases 90% to 95%, respectively, but did not affect the isolated porcine submaxillary gland N-acetylgalactosaminyltransferase. Benzyl alcohol did not inhibit the milk galactosyltransferase incorporated into DMPC or DPPC liposomes but showed a complex effect on the activity of the enzyme incorporated into DSPC vesicles, a stimulation of activity at low concentrations followed by an inhibition. A lipid environment consisting of saturated lipids appears to present a barrier to inhibiting substances such as linoleic acid and benzyl alcohol, or lipid may stabilize the active conformation of the enzyme. The different effects of these reagents on four transferases of the Golgi complex suggest that the lipid environment around these enzymes may be different for each transferase.

Biosynthesis of hyaluronic acid in the vitreous V. Studies on a particulate hyalocyte glycosyl transferase

Previous studies on the in vitro, enzymatic biosynthesis of hyaluronic acid by extracts of calf vitreous hyalocytes have concentrated on the properties of a soluble glycosyl transferase. The present report describes the properties of a particulate glycosyl transferase from hyalocytes and its role in hyaluronic acid biosynthesis. The enzyme catalyzes the incorporation of radioactivity from UDP-[ 14C]glucuronic acid (UDP-[ 14C]GlcUA) and UDP-[ 3H] N-acetylglucosamine (UDP-[ 3H]GlcNAc) into high molecular weight soluble glycosaminoglycan which appears in the reaction medium. In addition, radioactive material is removed from the denatured enzyme by treatment with 1 m-NaCl, 7 m-urea, and leech hyaluronidase. Pronase treatment also solubilizes bound radioactive material. It is proposed that the tightly bound material, solubilized by Pronase or hyaluronidase, may represent the initial formation of enzyme-bound hyaluronic acid, which is subsequently released into the medium following further polymerization.

Biosynthesis of hyaluronic acid in the vitreous VI. Isolation of a complex containing hyaluronic acid and glycosyl transferase activity and studies on the activity of a soluble glycosyl transferase

Addition of protamine sulfate to calf vitreous, after removal of hyalocytes, results in the formation of an insoluble complex of hyaluronic acid and protamine. The hyaluronic acid-protamine (HA-P) complex was found to possess glycosyl transferase activity, as shown by the incorporation of radioactivity from UDP-[ 14C]glucuronic acid and UDP-[ 14C] N-acetyl-glucosamine into the hyaluronic acid present in the complex. The incorporation of radio-activity was stimulated by the addition of Mg 2+, and for optimal incorporation both nucleotide sugar precursors of hyaluronic acid were required. When the HA-P complex is dissolved in 0·5-1 m-NaCl solutions, the glycosyl transferase can be separated from protamine, thereby regenerating a soluble system for the study of hyaluronic acid biosynthesis. The present report also describes the results of experiments designed to determine which agents affect the activity and heat stability of a soluble glycosyl transferase, extracted from hyalocytes, which similarly catalyzes hyaluronic acid synthesis. Treatment of the enzyme with N-acetylimidazole results in an inhibition of enzyme activity. When the treatment was carried out in the presence of substrates, no protection against the inhibition was observed. The enzyme was also inhibited by UTP and UDP, suggesting the role of this portion of the nucleotide sugar substrates in binding to the enzyme. Treatment with urea caused the enzyme to be inactivated by heat either in the presence or absence of endogeneous hyaluronic acid.

Occurrence of soluble glycosyltransferases in human amniotic fluid

Human amniotic fluid obtained by amniocentesis during the third trimester of pregnancy was found to contain glycosyltransferases for the transfer of galactose, N-acetylgalactosamine, N-acetylglucosamine and sialic acid from their nucleotide derivatives to various exogenous protein and small molecular weight acceptors. The specific activity of the galactosyl- and N-acetylgalactosaminyl transferases was found to be 30 to 40 times higher in amniotic fluid as compared to serum. The specific activity of N-acetylglucosaminyl- and sialyl transferases was only 3 to 6 fold higher in amniotic fluid.

UDP-N-acetylgalactosamine:Protein N-acetylgalactosaminyl transferase activity of human sera

A soluble glycosyltransferase which transfers N-acetylgalactosamine from UDP-N-acetylgalactosamine to exogenous protein acceptors is present in human serum. The proteins capable of accepting the hexosamine are basic myelin proteins from beef brain and spinal cord, and bovine submaxillary mucin from which sialic acid and hexosamine have been removed. Other proteins studied do not serve as acceptors. Alkaline borohydride treatment of the labeled protein acceptors releases over 80% of the radioactivity as N-acetylgalactosaminitol thus indicating that the galactosaminylation takes place primarily at the hydroxyaminoacids in these proteins. The enzyme is present in the sera of all individuals, irrespective of their ABO blood group. In this respect it differs from the galactosaminyl transferase that is presumed to be responsible for blood group A antigenicity.