NMR study of a Lewis(X) pentasaccharide derivative: solution structure and interaction with cations.

The structure and conformation of the synthetic pentasaccharide Gal(beta 1-4){Fuc(alpha 1-3)}GlcNAc(beta 1-3)Gal(beta 1-4)Glc-beta OMe of the Lewis(X) family has been determined by NMR spectroscopy in dimethyl sulfoxide and methanol. In these solvents, the binding constants with calcium have been evaluated as 9.5 and 29.6 M-1, respectively. Study of the interaction sites has been achieved through the use of paramagnetic divalent cations and distance triangulation methods. Two regions have been found, the first one in the vicinity of the fucose unit, the second one closer to the lactose part.     

Production of a complement inhibitor possessing sialyl Lewis X moieties by in vitro glycosylation technology

Recombinant soluble human complement receptor type 1 (sCR1) is a highly glycosylated glycoprotein intended for use as a drug to treat ischemia-reperfusion injury and other complement-mediated diseases and injuries. sCR1-sLe(x) produced in the FT-VI-expressing mutant CHO cell line LEC11 exists as a heterogeneous mixture of glycoforms, a fraction of which include structures with one or more antennae terminated by the sialyl Lewis X (sLe(x)) [Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc]) epitope. Such multivalent presentation of sLe(x) was shown previously to effectively target sCR1 to activated endothelial cells expressing E-selectin. Here, we describe the use of the soluble, recombinant alpha2-3 sialyltransferase ST3Gal-III and the alpha1-3 fucosyltransferase FT-VI in vitro to introduce sLe(x) moieties onto the N-glycan chains of sCR1 overexpressed in standard CHO cell lines. The product (sCR1-S/F) of these in vitro enzymatic glycan remodeling reactions performed at the 10-g scale has approximately 14 N-glycan chains per sCR1 molecule, comprised of biantennary (90%), triantennary (8.5%), and tetraantennary (1.5%) structures, nearly all of whose antennae terminate with sLe(x) moieties. sCR1-S/F retained complement inhibitory activity and, in comparison with sCR1-sLe(x) produced in the LEC11 cell line, contained twice the number of sLe(x) moieties per mole glycoprotein, exhibited a twofold increase in area under the intravenous clearance curve in a rat pharmacokinetic model, and exhibited a 10-fold increase in affinity for E-selectin in an in vitro binding assay. These results demonstrate that in vitro glycosylation of the sCR1 drug product reduces heterogeneity of the glycan profile, improves pharmacokinetics, and enhances carbohydrate-mediated binding to E-selectin.     

Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigen-containing glycoprotein

Glycoproteins constitute a class of compounds of increasing importance for pharmaceutical applications. The manipulation of bacterial protein glycosylation systems from Gram-negative bacteria for the synthesis of recombinant glycoproteins is a promising alternative to the current production methods. Proteins carrying Lewis antigens have been shown to have potential applications for the treatment of diverse autoimmune diseases. In this work, we developed a mixed approach consisting of in vivo and in vitro steps for the synthesis of glycoproteins containing the Lewis x antigen. Using glycosyltransferases from Haemophilus influenzae, we engineered Escherichia coli to assemble a tetrasaccharide on the lipid carrier undecaprenylphosphate. This glycan was transferred in vivo from the lipid to a carrier protein by the Campylobacter jejuni oligosaccharyltransferase PglB. The glycoprotein was then fucosylated in vitro by a truncated fucosyltransferase from Helicobacter pylori. Diverse mass spectrometry techniques were used to confirm the structure of the glycan. The strategy presented here could be adapted in the future for the synthesis of diverse glycoproteins. Our experiments demonstrate that bacterial enzymes can be exploited for the production of glycoproteins carrying glycans present in human cells for potential therapeutic applications.     

A convergent strategy for the preparation of N-glycan core di-, tri-, and pentasaccharide thioaldoses for the site-specific glycosylation of peptides and proteins bearing free cysteines

Mammalian glycoprotein biosynthesis produces heterogeneous ranges of proteins that possess the same peptide backbone but differ in the nature and site of glycosylation. This feature has frustrated efforts to develop therapeutic glycoproteins as well as the elucidation of biological functions of individual glycoforms. We have developed an attractive approach to well-defined glycoforms of glycoproteins by oxidative coupling of thioaldoses to cysteine-containing peptides and proteins to give disulfide-linked neoglycoconjugates. To this end, the chemical synthesis di-, tri-, and pentasaccharide N-glycan thioaldoses was undertaken. A convergent approach was used for the preparation of the pentasaccharide containing a 'synthetically difficult' beta-mannoside linkage. This linkage was installed by forming initially the corresponding beta-glucoside-containing pentasaccharide, followed by inversion of configuration at C-2. This approach exploited a levulonyl ester at C-2 of a glucosyl donor, which directed the coupling to give the beta-glucoside exclusively and could be removed selectively using hydrazine acetate without affecting other base-labile functionalities. The resulting alcohol was converted into a triflate, which was displaced by tri-n-butylammonium acetate to give a beta-mannosidic linkage. The trisaccharide N-glycan was prepared in a similar manner. Thioaldoses were prepared by displacing the peracetylated alpha-glycosyl chlorides with thioacetate to give the peracetylated beta-thioacetates, which upon saponification gave the desired compounds. The incubation of molar excesses of chitobiose thioaldose with cysteine-containing glutathione and BSA resulted in the site-specific formation of a disulfide-linked neoglycopeptide and neoglycoprotein, respectively.     

phi X174-directed DNA and protein syntheses in infected minicells.

Phi X174-infected minicells, produced by Escherichia coli PC2251, synthesized 11 phi X174-encoded polypeptides. The infecting single-stranded viral genome was converted to a double-stranded, closed circular, replicative form (replicative form I). Little, if any, replicative form I replication took place, and synthesis of progeny single-stranded molecules could not be detected.     

Development of a modified vitrification strategy suitable for subsequent scale-up for hepatocyte preservation

This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VS_basic (40% (v/v) ethylene glycol 0.6 M sucrose, i.e. 7.17 M ethylene glycol 0.6 M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VS_basic and increase the solution’s total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5 days.Results indicated Ficoll as the least toxic additive. Within 60 min, the exposure of hepatocytes to a solution composed of 9% Ficoll + 0.6 M sucrose (10⁻³ M Ficoll + 0.6 M sucrose) sustained attachment efficiency of 95%, similar to control. Furthermore, this additive did not cause any detriment to the attachment of these cells when supplementing the base vitrification solution VS_basic. The addition of 9% Ficoll, raised the total solute concentration to 74.06% (w/v) with a negligible 10⁻³ M increase in molarity of the solution. This suggests main factor in inducing detriment to cells was the molar contribution of the additive.Vitrification protocol for scale-up condition sustained hepatocyte suspension attachment efficiency and albumin production. We conclude that although established approach will permit scaling-up of vitrification of hepatocyte suspension, vitrification of hepatocytes which are attached prior to vitrification is more effective by comparison.     

Development of a modified vitrification strategy suitable for subsequent scale-up for hepatocyte preservation

This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VS_basic (40% (v/v) ethylene glycol 0.6 M sucrose, i.e. 7.17 M ethylene glycol 0.6 M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VS_basic and increase the solution’s total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5 days.     

Conformational studies of Lewis X and Lewis A trisaccharides using NMR residual dipolar couplings.

The conformations of the histo-blood group carbohydrate antigens Lewis X (Le(x)) and Lewis A (Le(a)) were studied by NMR measurements of one-bond C-H residual dipolar couplings in partially oriented liquid crystal solutions. A strategy for rapid calculation of the difference between theoretical and experimental dipolar couplings of a large number of model structures generated by computer simulations was developed, resulting in an accurate model structure for the compounds. Monte Carlo simulations were used to generate models for the trisaccharides, and orientations of each model were sought that could reproduce the experimental residual dipolar coupling values. For both, Le(a) and Le(x), single low energy models giving excellent agreement with experiment were found, implying a compact rigidly folded conformation for both trisaccharides. The new approach was also applied to the pentasaccharides lacto-N-fucopentaose 2 (LNF-2) and lacto-N-fucopentaose 3 (LNF-3) proving its consistency and robustness. For describing the conformation of tightly folded oligosaccharides, a definition for characterization of ring planes in pyranoside chairs is proposed and applied to the analysis of the relation between the fucose and galactose residues in the epitopes, revealing the structural similarity between them.     

Application and limitations of the methyl imidate protection strategy of N-acetylglucosamine for glycosylations at O-4: synthesis of Lewis A and Lewis X trisaccharide analogues

We describe here the synthesis of the allyl Le^a trisaccharide antigen as well as that of an analogue of the Le^x trisaccharide antigen, in which the galactose residue has been replaced by a glucose unit. Although successful fucosylations at O-4 of N-acetylglucosamine acceptors have been reported using perbenzylated thioethyl fucosyl donors under MeOTf activation, such conditions led in our case to the conversion of our acceptor to the corresponding alkyl imidates. Indeed, in this synthesis of the Le^a analogue, we demonstrate that the temporary protection of the N-acetyl group as a methyl imidate is advantageous to fucosylate at O-4. In contrast, we report here that glucosylation at O-4 of an N-acetylglucosamine monosaccharide acceptor using the α-trichloroacetimidate of peracetylated glucopyranose as a donor proceeded in better yields under activation with excess BF₃·OEt₂ than that of the corresponding methyl imidate. Therefore, we conclude that activation of thioglycoside donors by MeOTf to glycosylate at O-4 of a glucosamine acceptor is best accomplished following the temporary protection of the N-acetyl group as a methyl imidate, especially when the donors are highly reactive and prone to degradation. In contrast, if donor and acceptor can withstand multiple equivalents of BF₃·OEt₂, glycosylations at O-4 of a glucosamine acceptor with a trichloroacetimidate donor does not benefit from the temporary protection of the N-acetyl group as a methyl imidate.     

Characterization of a diagnostic Fab fragment binding trimeric Lewis X

Lewis X trisaccharides normally function as essential cell–cell interaction mediators. However, oligomers of Lewis X trisaccharides expressed by the parasite Schistosoma mansoni seem to be related to its evasion of the immune response of its human host. Here we show that monoclonal antibody 54‐5C10‐A, which is used to diagnose schistosomiasis in humans, interacts with oligomers of at least three Lewis X trisaccharides, but not with monomeric Lewis X. We describe the sequence and the 2.5 Å crystal structure of its Fab fragment and infer a possible mode of binding of the polymeric Lewis X from docking studies. Our studies indicate a radically different mode of binding compared to Fab 291‐2G3‐A, which is specific for monomeric Lewis X, thus providing a structural explanation of the diagnostic success of 54‐5C10‐A. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

The biosynthesis of Lewis X in Helicobacter pylori

The biosynthesis of the Lewis X determinant (Galß1-4[Fuc     

A novel strategy for production of a highly expressed recombinant protein in an active form.

Under standard growth conditions, E. coli transformed with the high-level expression vector pMON5525 produces recombinant DMAPP/AMP transferase in inactive, insoluble complexes. We have produced large amounts of active, soluble protein by growing and inducing the cells under osmotic stress in the presence of sorbitol and glycyl betaine. This caused an increase of up to 427-fold in the active yield, and the disappearance of the protein from the pelletable fraction of cell extracts. This treatment may have wide applicability.     

Latent fluorophores based on a self-immolative linker strategy and suitable for protease sensing

The self-immolative spacer para-aminobenzyl alcohol (PABA) was used as a key component in the design of new protease-sensitive fluorogenic probes whose parent phenol-based fluorophore is released through an enzyme-initiated domino reaction. First, the conjugation of the phenylacetyl moiety to 7-hydroxycoumarin (umbelliferone) and 7-hydroxy-9 H-(9,9-dimethylacridin-2-one) (DAO) by means of the heterobifunctional PABA linker has led to pro-fluorophores 6a and 6d whose enzyme activation by penicillin amidase was demonstrated. The second part of this study was devoted to the extension of this latent fluorophore strategy to the caspase-3 protease, a key mediator of apoptosis in mammalian cells. Fluorogenic caspase-3 substrates 11 and 13 derived from umbelliferone and DAO, respectively, were prepared. It was demonstrated that pro-fluorophore 11 is a sensitive fluorimetric reagent for the detection of this cysteine protease. Furthermore, in vitro assays with fluorogenic probe 13 showed a deleterious effect of biological thiols on fluorescence of the released acridinone fluorophore DAO that, to our knowledge, had not been reported until now.     

A solid-phase synthesis for beta-turn mimetics of sialyl Lewis X

A solid-phase synthesis of heterocyclic beta-turn mimetics of sialyl Lewis X, which is a natural carbohydrate ligand of selectins, was established. This synthetic method could be very useful for drug discovery of selectin antagonists using combinatorial chemistry techniques.     

Development of a sensitive separation and quantification method for sialyl Lewis X and Lewis X involving anion-exchange chromatography: biochemical characterization of alpha1-3 fucosyltransferase-VII

The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe(x)) in human leukocytes is mediated by alpha1-3 fucosyltransferase-VII (FucT-VII), which catalyzes the transfer of fucose from GDP-beta-fucose to the 3-OH of alpha2-3 sialyl N-acetyllactosamine (SA-LN). We developed a simple method for quantitating the reaction product of FucT-VII involving Anion-Exchange Chromatography (AEC). The AEC assay involved the separation of a radio-labeled acceptor from the unreacted nucleotide sugars with 0-0.5 M NH(4)OAc (pH9.0) on QAE-Toyopearl 550C. Furthermore, this assay enabled the separation of the fucosylated products of sialylated and non-sialylated oligosaccharides with this column. Analysis of the FucT-VI reaction mixture showed that Lewis X (Le(x)) was eluted in the flow-through fraction and sLe(x) was eluted with 0.1 M NH(4)OAc, and these products were clearly separated from the fraction of unreacted GDP-[(3)H]fucose. Therefore, this method could be a powerful tool for the characterization of recombinant FucT-VII and for establishing a high-throughput screening system for FucT-VII inhibitors. Beside FucT-VII, this method will be applicable to the assaying of many different glycosyltransferases, including sialyltransferases and glucosaminyltransferases, which are reactive to alpha2-3 SA-LN or N-acetyllactosamine sequences.     

Complete extraction in a form suitable for liquid scintillation counting of tritium-labeled proteins from polyacrylamide gels

Large (200 mm3) slices of polyacrylamide gels crosslinked with N,N'-diallyltartardiamide which contain tritium-labeled protein are readily solubilized in periodic acid for liquid scintillation counting of radioactivity, but the apparent recovery of label never exceeds 82%. Extraction of the slices with two commercial solubilizers at 60 degrees C gave recoveries of 82-90% which were not improved by prolonged incubation. Treatment of the slices at ambient temperature with 1.0 ml of 2% sodium periodate for 30 min followed by the addition of 0.7 ml of aqueous tetrabutylammonium hydroxide (40% w/v) gives solutions which can be immediately counted at 35% efficiency with low background and with 100% recovery of tritiated protein     

Highly regioselective synthesis of a 3-O-sulfonated arabino Lewis(a) asparagine building block suitable for glycopeptide synthesis

Using the stannylene method, the trisaccharide 2-acetamido-3-O-[6-O-benzyl-beta-D-galactopyranosyl]-4-O-[2,3,4-tri-O-benzyl-beta-D-arabinopyranosyl]-6-O-benzyl-2-deoxy-beta-D-glucopyranosyl azide was regioselectively sulfonated and, after reduction of the anomeric azide, coupled to Fmoc alpha-allyl aspartate. After Pd(0)-catalyzed deallylation, the sulfatyl Lewis(a) asparagine building block was obtained, suitable for solid-phase glycopeptide synthesis applying the fluoride labile PTMSEL linker system.