Synthesis of New Pseudonucleosides Containing Chiral Cyclosulfamides as Agycone

Abstract

A series of chiral cyclosufamides have been synthesized in four steps, starting from N-benzoylaminoacids. Regioselective glycosylation of these pseudopyrimidic heterocycles was carried out after deprotection. Best glycosylation results were obtained by preliminary silylation of cyclosulfamides, and their condensation with a tetraacetylribofuranose and pentaacetylglucopyranose is described, which yielded the pseudonucleosides in a β-anomeric configuration.     

First total synthesis of a pentasaccharide repeating unit of the <Emphasis Type="Italic">O</Emphasis>-antigen of <Emphasis Type="Italic">Hafnia alvei</Emphasis> PCM 1529

A pentasaccharide repeating unit of the O-antigen of Hafnia alvei has been synthesized in a concise manner. High yielding glycosylation steps and minimum number of protecting group manipulation steps are the key features of this synthesis. A two-step, one-pot phase transfer oxidation protocol has been applied for the preparation of d-galacturonic acid. C.D.R.I. communication no. 7245.     

Application of a chemoenzymatic glycosylation method to α-chymotrypsin and Candida rugosa lipase surface modifications

A recently developed chemoenzymatic glycosylation procedure has been successfully applied on two hydrolytic enzymes, α-chymotrypsin and Candida rugosa lipase. First, a number of sucrose molecules have been bound to the surface lysine residues and then, lengthening of the glycosidic chains has been carried out by the action of a levansucrase from Bacillus subtilis. For both steps, reaction conditions have been studied in order to obtain a range of glycosylation degrees. The influence of glycoside binding on biocatalyst surface characteristics has been assessed and a progressive increase in global enzyme hydrophilic character with glycosylation has been observed. Besides, the study of hydrolytic activity and kinetic constants showed that the performed modifications brought about a certain decrease in enzyme hydrolytic activity and very slight variations in enzyme-substrate affinity.     

Insights in the glycosylation steps during biosynthesis of the antitumor anthracycline cosmomycin: characterization of two glycosyltransferase genes

Glycosylation pattern in cosmomycins is a distinctive feature among anthracyclines. These antitumor compounds possess two trisaccharide chains attached at C-7 and C-10, each of them with structural variability, mainly at the distal deoxysugar moieties. We have characterized a 14-kb chromosomal region from Streptomyces olindensis containing 13 genes involved in cosmomycin biosynthesis. Two of the genes, cosG and cosK, coding for glycosyltransferase were inactivated with the generation of five new derivatives. Structural elucidation of these compounds showed altered glycosylation patterns indicating the capability of both glycosyltransferases of transferring deoxysugars to both sides of the aglycone and the flexibility of CosK with respect to the deoxysugar donor. A model is proposed for the glycosylation steps during cosmomycins biosynthesis.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Contribution of processing events to the molecular heterogeneity of four banding types of phaseolin,the major storage protein of Phaseolus vulgaris L.

Summary The origin of the molecular heterogeneity of phaseolin was investigated by studying, both in vivo and in vitro, the synthesis and processing of four different banding types of phaseolin in five cultivars of Phaseolus vulgaris L. The results demonstrate: I) Newly-synthesized (unprocessed) phaseolin in all cultivars is composed of three major components. These differ between cultivars, both in charge and Mr. II) The processing of these precursors is highly conserved and consists of the co-translational cleavage of a signal peptide, two glycosylation steps in the endoplasmic reticulum and a further modification inside the protein bodies to give the mature form. III) Some of the molecular heterogeneity of each phaseolin banding type is due to a different extent of glycosylation of its polypeptide components.     

The OST‐complex as target for RNAi‐based pest control in Nilaparvata lugens

RNAi‐based pest control strategies are emerging as environment friendly and species‐specific alternatives for the use of conventional pesticides. Because N‐glycosylation is important for many biological processes, such as growth and development, the early steps of protein N‐glycosylation are promising targets for an RNAi‐based pest control strategy. Through injection of dsRNAs, the expression of the catalytic subunits of the oligosaccharyl transferase complex was efficiently silenced in nymphs of the notorious rice pest insect Nilaparvata lugens. Silencing of both STT3 isoforms resulted in a high mortality of the N. lugens nymphs. However, our data reveals the occurrence of a functional redundancy between the two isoforms when silencing only one of the isoforms. These observations confirm the potential to use the early genes in the N‐glycosylation pathway as targets for an RNAi‐based pest control strategy. In addition, the existence of a functional redundancy between the two STT3 isoforms presents a factor which one must take into account when designing RNAi‐based approaches.     

LEW3, encoding a putative α‐1,2‐mannosyltransferase (ALG11) in N‐linked glycoprotein,plays vital roles in cell‐wall biosynthesis and the abiotic stress response in Arabidopsis thaliana

N‐linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N‐linked glycosylation shares a common pathway with assembly of a dolichol‐linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an α‐1,2‐mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol‐linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N‐glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low‐level accumulation of Man₃GlcNAc₂ and Man₄GlcNAc₂ glycans, structures that are seldom detected in wild‐type plants. In addition, the lew3 mutant has low levels of normal high‐mannose‐type glycans, but increased levels of complex‐type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N‐glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild‐type. These results demonstrate that protein N‐glycosylation plays crucial roles in plant development and the response to abiotic stresses.     

The efficient total synthesis of bis-glycosyl apigenin from naringenin: a greener way

An efficient total synthesis of 7-O-β-d-glucopyranosyl-4′-O-α-l-rhamnopyranosyl apigenin (1) was developed in only four steps from naringenin. Compared with our previously reported first total synthesis route (six steps and 19.6% overall yield), this new route contained two steps of highly regioselective glycosylation without any selective protection steps. 7,4′-di-O-β-d-glucopyranosyl apigenin (2) was also prepared efficiently by this method. The method is environmentally friendly, economical, and provides a greener method for flavonoid synthesis starting from an inexpensive flavanone.     

Neutral N-glycan patterns of the gastropods Limax maximus, Cepaea hortensis, Planorbarius corneus, Arianta arbustorum and Achatina fulica

The N-glycosylation potentials of Limax maximus, Cepaea hortensis, Planorbarius corneus, Arianta arbustorum and Achatina fulica were analysed by investigation of the N-glycan structures of the skin and viscera glycoproteins by a combination of HPLC and mass-spectrometry methods. It is one of the first steps to enlarge the knowledge on the glycosylation abilities of gastropods, which may help to establish new cell culture systems, to uncover new means for pest control for some species, and to identify carbohydrate-epitopes which may be relevant for immune response. All snails analysed contained mainly oligomannosidic and small paucimannosidic structures, often terminated with 3-O-methylated mannoses. The truncated structures carried modifications by β1-2-linked xylose to the β-mannose residue, and/or an α-fucosylation, mainly α1,6-linked to the innermost N-acetylglucosaminyl residue of the core. Many of these structures were missing the terminal N-acetylglucosamine, which has been shown to be a prerequisite for processing to complex N-glycans in the Golgi. In some species (Planorbarius corneus and Achatina fulica) traces of large structures, terminated by 3-O-methylated galactoses and carrying xylose and/or fucose residues, were also detected. In Planorbarius viscera low amounts of terminal α1-2-fucosylation were determined. Combining these results, gastropods seem to be capable to produce all kinds of structures ranging from those typical in mammals through to structures similar to those found in plants, insects or nematodes. The detailed knowledge of this very complex glycosylation system of the gastropods will be a valuable tool to understand the principle rules of glycosylation in all organisms.     

Easy Production of a Glycolipid Analogue Using Animal Cells in Culture

A glycolipid analogue, GM4‐type ganglioside, was obtained by a combination of chemical synthesis and biosynthetic processes in animal cells with dodecyl β‐D ‐galactoside (Gal C12) as primer. The primer was conveniently prepared in two steps: glycosylation, followed by deacetylation. The primer was introduced to mouse melanoma B16 cells to serve as substrate for cellular, enzyme‐catalyzed glycosylation. Incubation of the cells in the presence of the primer resulted in sialylation of the galactose residue to afford a GM4 analogue that was released from the cells to the culture medium. The strategy of preparation of the GM4 analogue described in this study is a viable alternative to the existing methods. The saccharide‐primer method is fast, convenient, not requiring expensive enzymes and glycosyl donors, and highly stereoselective.     

Elevated Golgi pH impairs terminal N‐glycosylation by inducing mislocalization of Golgi glycosyltransferases

Acidic pH of the Golgi lumen is known to be crucial for correct glycosylation, transport and sorting of proteins and lipids during their transit through the organelle. To better understand why Golgi acidity is important for these processes, we have examined here the most pH sensitive events in N‐glycosylation by sequentially raising Golgi luminal pH with chloroquine (CQ), a weak base. We show that only a 0.2 pH unit increase (20 µM CQ) is sufficient to markedly impair terminal α(2,3)‐sialylation of an N‐glycosylated reporter protein (CEA), and to induce selective mislocalization of the corresponding α(2,3)‐sialyltransferase (ST3) into the endosomal compartments. Much higher pH increase was required to impair α(2,6)‐sialylation, or the proximal glycosylation steps such as β(1,4)‐galactosylation or acquisition of Endo H resistance, and the steady‐state localization of the key enzymes responsible for these modifications (ST6, GalT I, MANII). The overall Golgi morphology also remained unaltered, except when Golgi pH was raised close to neutral. By using transmembrane domain chimeras between the ST6 and ST3, we also show that the luminal domain of the ST6 is mainly responsible for its less pH sensitive localization in the Golgi. Collectively, these results emphasize that moderate Golgi pH alterations such as those detected in cancer cells can impair N‐glycosylation by inducing selective mislocalization of only certain Golgi glycosyltransferases. J. Cell. Physiol. 220: 144–154, 2009. © 2009 Wiley‐Liss, Inc.     

Expression, Purification, and Bioassay of Human Stanniocalcin from Baculovirus-Infected Insect Cells and Recombinant CHO Cells

Stanniocalcin is a calcium- and phosphate-regulating glycoprotein hormone that was first described in fish where it functions in preventing hypercalcemia. Human cDNA clones encoding the homolog of stanniocalcin have been recently isolated. In this study, the full-length cDNA coding for human stanniocalcin (hSTC) was cloned into both baculovirus and CHO expression vectors. Recombinant hSTC was then produced efficiently from both baculovirus-infected insect cells and CHO cells in large-scale bioreactors. Purification protocols were developed and used to purify recombinant hSTC from both sources in four chromatography steps. The hSTCs from both expression systems were secreted as glycosylated proteins and as disulfide-linked homodimers. The results from glycosylation studies indicated that stanniocalcin from both sources contained N-linked oligosaccharides but no O-linked sugars. In anin vivobioassay based on the inhibition of gill calcium transport in fishes, the baculovirus and CHO-expressed protein showed biological activity which is dose dependent. The inhibitory effects of hSTC produced from both systems were essentially equipotent in fishes, despite the differences in glycosylation. Consequently, the precise role of the carbohydrate moiety in recombinant hSTC remains to be determined.     

Spatiotemporal metabolic regulation of anthocyanin and related compounds during the development of marginal picotee petals in <Emphasis Type="Italic">Petunia hybrida</Emphasis> (Solanaceae)

Structures and levels of anthocyanin-related compounds were analyzed during the development of marginal picotee petals in white-center and white-marginal cultivars of Petunia hybrida. In the white site of a white-center cultivar, higher concentrations of quercetin derivatives possessing 7-O-glucoside and/or 3′-O-glucoside occurred than in the colored site, suggesting that these two quercetin glycosylation steps are site-specifically regulated. The boundary areas of petal coloration were composed of cells showing various color densities, whose uniformity among adjacent cells varied between these cultivars. These results indicate diversity in spatiotemporal regulation of anthocyanin biosynthesis and flavonol glycosylations between Petunia cultivars during marginal picotee formation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of <Emphasis Type="Italic">Saccharomyces cerevisiae ret1-1</Emphasis> mutation on glycosylation and localization of the secretome

To study the effect of the ret1-1 mutation on the secretome, the glycosylation patterns and locations of the secretory proteins and glycosyltransferases responsible for glycosylation were investigated. Analyses of secretory proteins and cell wall-associated glycoproteins showed severe impairment of glycosylation in this mutant. Results from 2D-polyacrylamide gel electrophoresis (PAGE) indicated defects in the glycosylation and cellular localization of SDS-soluble cell wall proteins. Localization of RFP-tagged glycosyltransferase proteins in ret1-1 indicated an impairment of Golgi-to retrograde transport at a non-permissive temperature. Thus, impaired glycosylation caused by the mislocalization of ER resident proteins appears to be responsible for the alterations in the secretome and the increased sensitivity to ER stress in ret1-1 mutant cells.     

Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N‐glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N‐glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed‐batch culture of a CHO cell line producing recombinant human interferon‐γ (IFN‐γ). Of the 24 N‐glycosylation genes examined, 21 showed significant up‐ or down‐regulation of gene expression as the fed‐batch culture progressed from exponential, stationary and death phase. As the fed‐batch culture progressed, there was also an increase in less sialylated IFN‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed‐batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N‐glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible “bottlenecks” or “compromised” pathways in N‐glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516–528. © 2010 Wiley Periodicals, Inc.     

Characterization of the <Emphasis Type="Italic">N</Emphasis>-glycosylation phenotype of erythrocyte membrane proteins in congenital dyserythropoietic anemia type II (CDA II/HEMPAS)

Congenital dyserythropoetic anemia type II (CDA II) is characterized by bi- and multinucleated erythroblasts and an impaired N-glycosylation of erythrocyte membrane proteins. Several enzyme defects have been proposed to cause CDA II based on the investigation of erythrocyte membrane glycans pinpointing to defects of early Golgi processing steps. Hitherto no molecular defect could be elucidated. In the present study, N-glycosylation of erythrocyte membrane proteins of CDA II patients and controls was investigated by SDS-Page, lectin binding studies, and MALDI-TOF/MS mapping in order to allow an embracing view on the glycosylation defect in CDA II. Decreased binding of tomato lectin was a consistent finding in all typical CDA II patients. New insights into tomato lectin binding properties were found indicating that branched polylactosamines are the main target. The binding of Aleuria aurantia, a lectin preferentially binding to α1-6 core-fucose, was reduced in western blots of CDA II erythrocyte membranes. MALDI-TOF analysis of band 3 derived N-glycans revealed a broad spectrum of truncated structures showing the presence of high mannose and hybrid glycans and mainly a strong decrease of large N-glycans suggesting impairment of cis, medial and trans Golgi processing. Conclusion: Truncation of N-glycans is a consistent finding in CDA II erythrocytes indicating the diagnostic value of tomato-lectin studies. However, structural data of erythrocyte N-glycans implicate that CDA II is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts.     

Production and purification of recombinant human granulocyte–macrophage colony stimulating factor (GM-CSF) from high cell density cultures of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Granulocyte–macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor, which has been used as a therapeutic agent in clinical cases like neutropenia. In this study, we report the production of recombinant human GM-CSF in the methylotrophic yeast Pichia pastoris through secretory expression using the inducible AOX1 promoter. Recombinant P. pastoris GS115 cells were grown in fed batch cultures to obtain a biomass density of 55.6 gDCW L⁻¹ and a high volumetric activity of 131 mg L⁻¹ of GM-CSF. The protein migrated as a diffuse band on SDS-PAGE at the range of 28–35 kDa indicating differential glycosylation. The secreted protein was purified to 95% in two steps using cation exchange and size exclusion chromatography.     

Proteomic identification of intracellular vesicle trafficking and protein glycosylation requirements for lumen inflation in Ciona notochord

Lumen formation and inflation are crucial steps for tubular organ morphogenesis, yet the underling mechanism remains largely unrevealed. Here, we applied 4D proteomics to screen the lumenogenesis-related proteins and revealed the biological pathways potentially that are involved in lumen inflation during notochord lumen formation in the ascidian Ciona savignyi. In total, 910 differentiated expressed proteins (DEPs) were identified before and after notochord lumen formation utilizing Mfuzz analysis. Those DEPs were grouped into four upregulated clusters based on their quantitative expression patterns; the functions of these proteins were enriched in protein metabolic and biosynthetic process, the establishment of localization, and vesicle-mediated transport. We analyzed the vesicle trafficking cluster and focused on several vesicle transport hub proteins. In vivo function-deficient experiments showed that mutation of vesicle transport proteins resulted in an abnormal lumen in notochord development, demonstrating the crucial role of intracellular trafficking for lumen formation. Moreover, abundant extracellular matrix proteins were identified, the majority of which were predicted to be glycosylated proteins. Inhibition of glycosylation markedly reduced the lumen expansion rate in notochord cells, suggesting that protein glycosylation is essential for lumenogenesis. Overall, our study provides an invaluable resource and reveals the crucial mechanisms in lumen formation and expansion.     

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Studies of protein N‐glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N‐glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N‐glycan patterns as documented using mass spectrometry and glycan‐recognising antibodies, indicating successful identification of null mutations in the target glyco‐genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3‐fucosyltransferase (Lj3fuct) mutant completely lacked α1,3‐core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N‐glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N‐acetylglucosaminyltransferase I, and α1,3‐fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N‐glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N‐glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian‐like N‐glycosylation features.     

Design,Synthesis, and Antiviral Evaluation of Some Polyhalogenated Indole C-nucleosides

2,5,6-Trichloro-1-(β-D-ribofuranosyl)benzimidazole (TCRB), 2-bromo-5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (BDCRB) and 2-benzylthio-5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (BTDCRB) are benzimidazole nucleosides that exhibit strong and selective anti-HCMV activity. Polyhalogenated indole C-nucleosides were prepared as 1-deaza analogs of the benzimidazole nucleosides TCRB and BDCRB. A mild Knoevenagel coupling reaction between an indol-2-thione and a ribofuranose derivative was developed for the synthesis of 2-benzylthio-5,6-dichloro-3-(β-D-ribofuranosyl)indole (12). 3-(β-D-ribofuranosyl)-2,5,6-trichloroindole (16) was prepared from 12 in 4 steps. A Lewis acid-mediated glycosylation method was then developed to prepare the targeted 2-haloindole C-nucleoside 16 stereoselectively in four steps from the corresponding 2-haloindole aglycons. Only 12 was active against HCMV but it also was somewhat cytotoxic.