Glycosidic Moiety Research Articles

Structure and Function of the Long Pentraxin PTX3 Glycosidic Moiety: Fine-Tuning of the Interaction with C1q and Complement Activation

The prototypic long pentraxin PTX3 is a unique fluid-phase pattern recognition receptor that plays a nonredundant role in innate immunity and female fertility. The PTX3 C-terminal domain is required for C1q recognition and complement activation and contains a single N-glycosylation site on Asn 220. In the present study, we characterized the structure of the human PTX3 glycosidic moiety and investigated its relevance in C1q interaction and activation of the complement classical pathway. By specific endo and exoglycosidases digestion and direct mass spectrometric analysis, we found that both recombinant and naturally occurring PTX3 were N-linked to fucosylated and sialylated complex-type sugars. Interestingly, glycans showed heterogeneity mainly in the relative amount of bi, tri, and tetrantennary structures depending on the cell type and inflammatory stimulus. Enzymatic removal of sialic acid or the entire glycosidic moiety equally enhanced PTX3 binding to C1q compared to that in the native protein, thus indicating that glycosylation substantially contributes to modulate PTX3/C1q interaction and that sialic acid is the main determinant of this contribution. BIAcore kinetic measurements returned decreasing K(off) values as sugars were removed, pointing to a stabilization of the PTX3/C1q complex. No major rearrangement of PTX3 quaternary structure was observed after desialylation or deglycosylation as established by size exclusion chromatography. Consistent with C1q binding, PTX3 desialylation enhanced the activation of the classical complement pathway, as assessed by C4 and C3 deposition. In conclusion, our results provided evidence of an involvement of the PTX3 sugar moiety in C1q recognition and complement activation.

Large-scale production of cellulose-binding domains. Adsorption studies using CBD-FITC conjugates

A method for the gram-scale production of cellulose-binding domains (CBD) through the proteolytic digestion of a commercial enzymatic preparation (Celluclast) was developed. The CBD obtained, isolated from Trichoderma reesei cellobiohydrolase I, is highly pure and heavily glycosylated. The purified peptide has a molecular weight of 8.43 kDa, comprising the binding module, a part of the linker, and about 30% glycosidic moiety. Its properties may thus be different from recombinant ones expressed in bacteria. CBDfluorescein isothiocyanate conjugates were used to study the CBD-cellulose interaction. The presence of fluorescent peptides adsorbed on crystalline and amorphous cellulose fibers suggests that amorphous regions have a higher concentration of binding sites. The adsorption is reversible, but desorption is a very slow process. Abbreviations: CBHI – Cellobiohydrolase I; CBHII – Cellobiohydrolase II; CBD – Cellulose-binding domain; CBDCBHI, CBDCBHII – Cellulose-binding domain of cellobiohydrolase I and II, respectively; FITC – Fluorescein isothiocyanate

Kinetics of bromhexine-mediated down-regulation of focal adhesive molecules of uterus and trophectoderm affecting conception in the rat

Purpose Quantitative evaluation of properties of bromhexine (B) for expression of uterine proteins in ovariectomized (OVX) and pregnant rats. Materials and Methods Expression of proteins through SDS-PAGE, along with incorporation of glycosidic moieties, was conducted in pregnant and OVX rats under B influence. These findings were corroborated with other tests such as implantation sites, fetal and litter sizes in pregnant rats. Results In OVX animals, even under the influence of estradiol dipropionate and progesterone, the B recreated a condition akin to OVX animals. It also induced 50–80% inhibition in the incorporation of glycosidic moieties to polypeptide chain. Distinct reduction in implantation sites, fetal sizes and interference in the conception (16/46) in pregnant rats substantiated the results of the action of B as an antiimplantation agent. Conclusion Bromhexine has shown interference in blastocyst attachment, conception, reduction in number of implantation sites and dwarfing of fetuses; hence, it is a potential candidate for antiimplantation.

Flavonols and an indole alkaloid skeleton bearing identical acylated glycosidic groups from yellow petals of Papaver nudicaule

From yellow petals of Iceland poppy, besides the known flavonoid gossypitrin, seven kaempferol derivatives were isolated. In addition to kaempferol 3- O-β-sophoroside and kaempferol 3- O-β-sophoroside-7- O-β-glucoside, known from other plants, the mono- and dimalonyl conjugates of the latter were identified by MS and NMR spectroscopy. Structure analyses of a set of co-occurring pigments, the nudicaulins, revealed that they have the identical acylated glycoside moieties attached to a pentacyclic indole alkaloid skeleton for which the structure of 19-(4-hydroxyphenyl)-10 H-1,10-ethenochromeno[2,3- b]indole-6,8,18-triol was deduced from MS and NMR as well as chemical and chiroptical methods.

Disposition of Selected Flavonoids in Fruit Tissues of Various Tomato (Lycopersicon esculentumMill.) Genotypes

Flavonoids have been studied extensively because they offer great potential health benefits. In this study, enzymatic hydrolysis of glycosylated quercetin, kaempferol, and naringin was used to obtain their sugar-free aglycones. The investigation also employed a validated HPLC method to obtain the chiral disposition of the aglycone naringenin enantiomers. These analyses were conducted on exocarp, mesocarp, and seed cavity tissues of field-grown tomato (Lycopersicon esculentum Mill.) mutants (anthocyanin absent, atroviolacea, and high pigment-1) and their nearly isogenic parent (cv. Ailsa Craig) at immature green, "breaker", and red ripe maturity stages. Concentrations of all flavonoids using enzymatic hydrolysis were significantly higher than previously reported concentrations using acid hydrolysis. Presumably, this occurred due to a more specific and rapid hydrolysis of the glycoside moiety by the beta-glucosidase enzyme. The glycoside S-naringin was the predominant enantiomer in all fruit tissues, although the aglycones free R- and S-naringenin were detected in both exocarp and mesocarp. Whereas there was significantly more quercetin than kaempferol in exocarp tissue, they were present in about equal concentrations in the mesocarp. Quercetin concentrations were higher in the exocarp and mesocarp of immature green and breaker fruit of the high pigment-1 mutant than in the other genotypes, supporting the observed photoprotection and potential health benefits of the high pigment-1 tomato genotype.

New Dinucleoside Analogues via Cross‐Coupling Metathesis.

Synthesis of 3′-3′, 5′-5′, and 3′-5′ dimeric thymidine, linked by an olefinic chain between glycosidic moieties is described. Cross metathesis reaction of 3′ or 5′ O-allyl analogues of thymidine led to the expected 3′-3′ and 5′-5′ dimeric compounds, respectively. In order to obtain the 3′-5′ dimer, 5′-O-allyl and 3′-O-allyl monomers were first linked by their free 3′ OH and 5′ OH groups through a glutaryl spacer; ring closing metathesis was then operated upon this temporary dimer, followed by glutaryl removal. #Financial support from the “Conseil Regional du Limousin” is gratefully acknowledged. The authors are grateful to Dr. Michel Guilloton for useful comments on the manuscript.

Human IgE Binding to the Glycosidic Moiety of Bovine κ-Casein

IgE ability for recognizing milk proteins was assayed in the serum of an adult atopic patient who outgrew cow milk allergy in early childhood. A number of protein species included in casein from bovine milk were detected by human IgE in immunoblotting experiments. Comparing these results with those obtained from an analysis using antibody preparations specifically directed toward the different casein fractions, IgE-reactive bands were identified as isoforms of kappa-casein. IgE-reactive protein was not present in neither bovine cheese, regardless of cheese-making technology and time ripening, nor milk from any other dairy animal, such as ewe, goat, and water buffalo. Chemical deglycosylation of protein bands immobilized onto nitrocellulose proved that the glycosidic moiety of bovine kappa-casein was principally involved in IgE recognition.

Isolation from Gluconacetobacter diazotrophicus cell walls of specific receptors for sugarcane glycoproteins, which act as recognition factors

Glycoproteins from sugarcane stalks have been isolated from plants field-grown by size-exclusion chromatography. Some of these glycoproteins, previously labelled with fluorescein isothiocyanate, are able to bind to the cell wall of the sugarcane endophyte, N 2-fixing Gluconacetobacter diazotrophicus, and largely removed after washing the bacterial cells with sucrose. This implies that sugarcane glycoproteins use β-(1 → 2)-fructofuranosyl fructose domains in their glycosidic moiety to bind to specific receptors in the bacterial cell walls. These receptors have been isolated by affinity chromatography on a sugarcane glycoprotein-agarose matrix, desorbed with sucrose and characterized by sodium dodecyl sulfate polyacrylamide gel electrophresisand capillary electrophoresis (CE).

Inhibition of Mitosis by Glycopeptide Dendrimer Conjugates of Colchicine

Glycopeptide dendrimers have been prepared bearing four or eight identical glycoside moieties at their surface (beta-glucose, alpha-galactose, alpha-N-acetyl-galactose, or lactose), natural amino acids within the branches (Ser, Thr, His, Asp, Glu, Leu, Val, Phe), 2,3-diaminopropionic acid as the branching unit, and a cysteine residue at the core. These dendrimers have been used as drug-delivery devices for colchicine. Colchicine was attached to the dendrimers at the cysteine thiol group through a disulfide or thioether linkage. The biological activities of the glycopeptide dendrimer conjugates were evaluated in HeLa tumor cells and non-transformed mouse embryonic fibroblasts (MEFs). The concentrations of glycopeptide dendrimer drug conjugates required to achieve inhibition of cell proliferation by interference with the tubulin system were found to be higher (IC50 > 1 microM) compared to the required colchicine concentration. On the other hand, the glycopeptide dendrimer conjugates inhibited the proliferation of HeLa cells 20-100 times more effectively than the proliferation of MEFs. In comparison, non-glycosylated dendrimers and colchicine itself showed a selectivity of 10-fold or less for HeLa cells.

Isolation of specific receptors from Xanthomonas albilineans cell walls for lectins-like glycoproteins of sugarcane

Two families of sugarcane glycoproteins differing in their molecular mass have been isolated from sugarcane stalks. These glycoproteins specifically bind to cell wall receptors of Xanthomonas albilineans, a sugarcane pathogen, producing bacterial agglutination. Bound glycoproteins can be desorbed from bacterial cell walls by galactitol, a component of the glycosidic moiety of the sugarcane protein. This indicates that sugarcane glycoproteins bind through their glycosidic rest to the peptide moiety of the bacterial receptor. Several cell wall receptors have been isolated by affinity chromatography and separated by capillary electrophoresis.

Purificação de lacases PPO-I de <em>Botryosphaeria rhodina

Laccases are glycoprotein polyphenol oxidases which are involved in fungal pathogenicity and they are also useful for biotechnological applications. The ligninolytic ascomycete, Botryosphaeria rhodina, has been studied as producer of exopolysaccharide and PPO-I and PPO-II laccases induced by veratryl alcohol. However, as the induced laccases have not been isolated, the aim of this study was to purify the enzyme and to identify the carbohydrates constituents of the glycosidic moiety. The fungus was cultivated on broth Vogel, 1% glucose and 30.4mM veratryl alcohol during 4.5 days at 28°C/180 rpm. The extracellular fluid showed high carbohydrate concentration and the stability of PPO-I laccase under conditions of refrigeration and freezing at 4ºC-18ºC over 40 days. The purification was developed by ultrafiltration using a NMWL 100 and 30 kDa membrane, gelfiltration on Sephadex G-100, and ion-exchange chromatography on DEAE-cellulose. The purified laccase was identified as a glycoprotein, weight molecular 113 kDa, consisting of 40% protein and 60% carbohydrate identified by HPAEC-PAD as fucose, galactose, mannose, glucose and glucosamine

New Dinucleoside Analogues via Cross-Coupling Metathesis#

Synthesis of 3′-3′, 5′-5′, and 3′-5′ dimeric thymidine, linked by an olefinic chain between glycosidic moieties is described. Cross metathesis reaction of 3′ or 5′ O-allyl analogues of thymidine led to the expected 3′-3′ and 5′-5′ dimeric compounds, respectively. In order to obtain the 3′-5′ dimer, 5′-O-allyl and 3′-O-allyl monomers were first linked by their free 3′ OH and 5′ OH groups through a glutaryl spacer; ring closing metathesis was then operated upon this temporary dimer, followed by glutaryl removal. #Financial support from the “Conseil Regional du Limousin” is gratefully acknowledged. The authors are grateful to Dr. Michel Guilloton for useful comments on the manuscript.

Ecdysteroid Glycosides: Identification, Chromatographic Properties, and Biological Significance

Ecdysteroid glycosides are found in both animals and plants. The chromatographic behavior of these molecules is characteristic, as they appear much more polar than their corresponding free aglycones when analyzed by normal-phase high-performance liquid chromatography (HPLC), whereas the presence of glycosidic moieties has a very limited (if any) impact on polarity when using reversed-phase HPLC. Biological activity is greatly reduced because the presence of this bulky substituent probably impairs the interaction with ecdysteroid receptor(s). 2-Deoxy-20-hydroxyecdysone 22-O-beta-D-glucopyranoside, which has been isolated from the dried aerial parts of Silene nutans (Caryophyllaceae), is used as a model compound to describe the rationale of ecdysteroid glycoside purification and identification.

Myxol and 4-Ketomyxol 2′-Fucosides, not Rhamnosides, from Anabaena sp. PCC 7120 and Nostoc punctiforme PCC 73102, and Proposal for the Biosynthetic Pathway of Carotenoids

We identified the molecular structures of carotenoids in some Anabaena and Nostoc species. The myxoxanthophyll and ketomyxoxanthophyll in Anabaena (also known as Nostoc) sp. PCC 7120, Anabaena variabilis IAM M-3, Nostoc punctiforme PCC 73102 and Nostoc sp. HK-01 were (3R,2'S)-myxol 2'-fucoside and (3S,2'S)-4-ketomyxol 2'-fucoside, respectively. The glycoside moiety of the pigments was fucose, not rhamnose. The major carotenoids were beta-carotene and echinenone, and the minor ones were beta-cryptoxanthin, zeaxanthin, canthaxanthin and 3'-hydroxyechinenone. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, we proposed a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. Since only zeta-carotene desaturase (CrtQ) from Anabaena sp. PCC 7120 and beta-carotene ketolase (CrtW) from N. punctiforme PCC 73102 have been functionally identified, the other genes were searched by sequence homology only from the functionally confirmed genes. Finally, we investigated the phylogenetic relationships among some Anabaena and Nostoc species, including some newly isolated species.

A new subfamily of fungal subtilases: structural and functional analysis of a Pleurotus ostreatus member

Pleurotus ostreatus produces several extracellular proteases which are believed to be involved in the regulation of the ligninolytic activities of this fungus. Recently, purification and characterization of the most abundant P. ostreatus extracellular protease (PoSl) have been reported. The sequence of the posl gene and of the corresponding cDNA has been determined, allowing the identification of its pre- and pro-sequences. A mature protein sequence has been verified by mass spectrometry mapping, the N-glycosylation sites have been identified and the glycosidic moieties characterized. Mature PoSl shows a cleaved peptide bond in the C-terminal region, which remains associated with the catalytic domain in a non-covalent complex. Reported results indicate that this enzyme is involved in the activation of other P. ostreatus secreted proteases, thus suggesting its leading role in cascade activation mechanisms. Analyses of the PoSl sequence by homology search resulted in the identification of a DNA sequence encoding a new protease, homologous to PoSl, in the Phanerochaete chrysosporium genome. A new subgroup of subtilisin-like proteases, belonging to the pyrolysin family, has been defined, which includes proteases from ascomycete and basidiomycete fungi.

Glycosilated Macromolecular Conjugates of Antiviral Drugs with a Polyaspartamide

Two new polymeric conjugates for specific liver targeting were prepared by conjugation of sugar moieties and antiviral drugs to α, β-poly[N-2-(hydroxyethyl)-dl-aspartamide] (PHEA). PHEA-galactopyranosylphenylthiocarbamide-mono-O-succinylganciclovir (conjugate 7) and PHEA-mannopyranosylphenylthiocarbamide-O-succinylacyclovir (conjugate 8) were synthesized according to a multi-step procedure which allowed for obtaining high product yield and process standardization. Conjugate 7 contained 7.5 and 8.5% of galactose and ganciclovir (substituent/repeating unit, mol/mol), respectively, and conjugate 8 contained 14.2 and 10.8% of mannose and acyclovir, respectively. In vitro studies demonstrated that both acyclovir and ganciclovir are released from the polymeric adducts at a release rate, which depended on the incubation medium. Though a detailed study evidenced that the two bioconjugates undergo different hydrolysis pathways, in both cases high drug release rate was found in plasma, while the glycosidic moiety was not released. Pharmacokinetic studies carried out by intravenous administration of the bioconjugates to Balb/c mice demonstrated that the conjugation of glycosidic moieties promotes the disappearance of the polymer from the bloodstream. The two derivatives displayed a different pharmacokinetic profile. In particular, the mannosyl conjugation promoted the rapid disposition of the macromolecule in the kidneys and in the liver, while prevented the accumulation in the spleen. On the contrary, the galactosyl derivative was found to dispose in the liver at the same extent of the naked polymer. Few considerations on the different behavior of the conjugates were reported.

Synthesis and Biological Evaluation of Oxindoles and Benzimidazolinones Derivatives.

AbstractFor Abstract see ChemInform Abstract in Full Text.

The Structure of Rigidoporus lignosus Laccase Containing a Full Complement of Copper Ions, Reveals an Asymmetrical Arrangement for the T3 Copper Pair

Laccase is a multicopper blue oxidase that couples the four-electron reduction of oxygen with the oxidation of a broad range of organic substrates, including phenols and arylamines. The enzyme is the object of intense biotechnological research, due to its employment in bioremediation of soils and water as well as in other biotechnological applications. We report here the cDNA and protein sequences, the post-translational modifications, the crystallization and X-ray structure determination of a laccase from the white-rot fungus Rigidoporus lignosus. The amino acid residues sequence deduced from cDNA clearly identified a pre-sequence of 21 residues representing the signal for extra-cellular localization. Mass spectrometry analysis performed on the salvage enzyme, confirmed the deduced sequence and precisely mapped two glycosylation sites at Asn337 and Asn435, determining the nature of the bound glycosidic moieties. The crystal structure was determined at 1.7A resolution from perfectly hemihedrally twinned crystals, by molecular replacement technique. While the overall structure closely resembled those reported for other fungal laccases, the analysis of the T2/T3 trinuclear cluster revealed an unprecedented coordination sphere for the T3 copper pair. No bridging oxygen ligand was present between the two T3 copper ions, which were no longer symmetrically coordinated. The observed structure could represent an intermediate along the process of four-electron reduction of oxygen to water taking place at the trinuclear copper cluster.

Anti-allergic activity of stilbenes from Korean rhubarb (Rheum undulatum L.): structure requirements for inhibition of antigen-induced degranulation and their effects on the release of TNF-α and IL-4 in RBL-2H3 cells

Stilbenes isolated from the rhizomes of Rheum undulatum (Korean rhubarb) and the related compounds were investigated on their anti-allergic activities. The results revealed that 3,5,4′-trimethylpiceatannol exhibited the most potent inhibition against β-hexosaminidase release as a marker of degranulation in RBL-2H3 cells with IC50 of 2.1μM, followed by trimethylresveratrol (IC50=5.1μM). Structural requirements of stilbenes for the activity are as follows: (1) The oxygen functions (–OCH3, –OH), especially methoxyl groups, are essential and their positions on aromatic rings are important for the activity; (2) the α–β double bond increased the activity; (3) the glycoside moiety dramatically decreased the activity; and (4) the substitution group at the 3′-position in trimethylresveratrol (3,5,4′-trimethoxystilbene) was preferably OH>H>OCH3 for the activity. Several active stilbenes (piceatannol, 3,5,4′-trimethylpiceatannol, resveratrol, trimethylresveratrol) also inhibited ionomycin-induced β-hexosaminidase release, suggesting that inhibition of Ca2+ influx or degranulation mechanisms after Ca2+ influx is important for their activities. Piceatannol, 3,5,4′-trimethylpiceatannol, resveratrol, and trimethylresveratrol also significantly inhibited antigen-induced release of TNF-α and IL-4 in RBL-2H3 cells.

New Triterpenoid Saponins from the Roots of Sinocrassula asclepiadea.

AbstractFor Abstract see ChemInform Abstract in Full Text.