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Cell surface carbohydrates in psoriasis. Defective cytoplasmic transport by glycoconjugates carrying fucose residues suggested by lectin staining.

Eleven biopsy specimens of normal skin and twenty-four biopsy specimens of psoriatic lesions were examined histochemically by using several lectins (Ulex europaeus, UEA-1; Dolichos biflorus, DBA; Bandeirea simplicifolia, BS-I; Concanvalia ensiformis, Con A; Triticum vulgaris, WGA; Ricinus communis, RCA; Arachis hypogoea, PNA) in order to evaluate the presence and distribution of various carbohydrates in normal and psoriatic keratinocytes. The findings revealed that keratinocytes from psoriatic lesions are distinguished by a different composition of carbohydrate residues incorporated in their plasma membranes. In particular, the intracellular transport of alpha-L-fucose, alpha-D-mannose, and alpha-D-glucose to the plasma cell membrane is impeded, whereas their synthesis in the cytoplasm of the psoriatic keratinocytes is largely unaltered. In addition, due to the lack of terminal alpha-L-fucose, the alpha-D-N-acetyl-galactosamine and alpha-D-galactose residues cannot be transferred to the plasma membranes and, therefore, the antigens for blood groups A and B remain incomplete in psoriatic epidermis. On the basis of these findings and in comparison with previous findings of our group on hyperproliferative, malignant keratinocytes, it is concluded that particularly the disordered cytoplasmic transport of alpha-L-fucose-carrying glycoconjugates may represent a specific defect in psoriasis, possibly linked with the pathogenesis of this disease.

Acetylgalactosamine↗

Quantitative determination of saccharide in Haemophilus influenzae type b glycoconjugate vaccines, alone and in combination with DPT, by use of high-performance anion-exchange chromatography with pulsed amperometric detection.

The stability and integrity of glycoconjugate vaccines requires determination of the total saccharide and quantification of the unbound or free saccharide present. The traditional assay for Hib conjugates, based on colorimetric determination of ribose, has been much improved by the use of base hydrolysis and analysis of the Hib subunit generated using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The production of this subunit was confirmed by NMR analysis. However, quantification of free Hib saccharide using this method was not possible in the combination vaccines evaluated due to interferences emanating from DPT. Thus a method based on TFA hydrolysis followed by the chromatographic separation and quantification of ribitol on a CarboPac MA1 column was developed. The method is selective, and with the use of ED40 electrode, requires only nanomole amounts for the chromatographic step, thereby ensuring that free saccharide can be monitored accurately in the formulated Hib-CRM vaccine alone and when in combination with other vaccines.

Carbohydrates↗

[Biological importance of the reactions between lectins and glycoconjugates in man].

Glucidic structures are now widely shown to be responsible of many recognition phenomenons. The biological efficiency of cellular membrane associated receptors depends on their stereospecific interactions with ligands. Glucidic structures are implicated in the interactions occurring between lectins and glycoconjugates. Although the knowledge about human lectins is still limited, such activities are now widely obvious for several cells: --erythrocytes: hemagglutin activity of glycophorin; --lymphocytes: Ts and NK cells; --platelets: plasma membrane and cell secreted lectins; --hepatic cells: lectins of hepatocytes and Kupffer cells; --muscular cells: galactose and heparin inhibitable lectins; --cells of human glomerular basement membrane: N-acetylosamines and N-acetyl neuraminic acid inhibitable lectins. To this list may be added the cytoagglutinating activity of fibronectin involved in cell-cell, cell-molecule or molecule-molecule interactions. The importance of lectinic agglutinins may be emphasized for several fundamental physiological mechanisms involved in molecular and cellular recognition: defenses against infection, coagulation, aging.

Adult↗

Pre- and postnatal expression of glycoconjugates (3-fucosyl-N-acetyllactosamine and HNK-1 epitopes) in the mouse inner ear.

The distribution of two glycoconjugates 3-fucosyl-N-acetyllactosamine (CD15) and HNK-1 epitope (CD57) in the inner ear of the NMRI mouse was analysed from the eighth day of gestation to the 16th day after birth. CD15 epitope distribution is developmentally regulated. The up- and down-regulation of expression, the change in the number of cells which are positive, the ingrowth of CD15-positive cells and their distribution, intracellular and/or cell-surface-associated expression, all assume a characteristic appearance at each developmental stage. Distribution of CD57 documented the nerve outgrowth and formation of the innervation of the vestibular apparatus and cochlear duct. Correlation between CD15 and CD57 expression patterns revealed differences in the interaction of the ingrowing fibres and epithelial tissue between the vestibular organ and the cochlea and differences in the development of the cristae and maculae.

Animals↗

Microbial glycoconjugates.

The surfaces of all microbes are 'sugar coated' with molecules such as lipopolysaccharides in Gram-negative bacteria, capsular polysaccharides in bacteria, lipoarabinomannans in mycobacteria and lipophosphoglycan in Leishmania. The basic structures of these glycoconjugates are known and, in the case of pathogens, they can function as virulence determinants. Recent publications have refined some of these structures and have elucidated interesting genes and proteins responsible for their biosynthesis.

Animals↗

Matrix-assisted laser desorption/ionization mass spectrometry of acidic glycoconjugates facilitated by the use of spermine as a co-matrix.

Negative-ion matrix-assisted laser desorption/ionization mass spectra of sialyated glycoconjugates were acquired employing 2,5-dihydroxybenzoic acid (DHB) in conjunction with spermine as a co-matrix. The addition of spermine to DHB permitted an improved crystal formation as well as a higher analyte solubility. Moreover, DHB/spermine appears to minimize alkali adduct formation, thus allowing the sample analysis without desalting. The combined matrix permitted the analysis of complex sialylated and sialylated/fucosylated structures down to the femotomole range. The ability to use such a matrix also facilitates determination of the sialic acid linkages (in combination with a specific enzyme cleavage). The matrix also appears suitable for studies on gangliosides.

Animals↗

Immunoferon, a glycoconjugate of natural origin, inhibits LPS-induced TNF-alpha production and inflammatory responses.

We have analyzed the effect of a patented glycoconjugate (GC) of natural origin, Inmunoferon, in the development of the response to endotoxemia induced by administration of LPS in rodents. We have observed that oral treatment with the drug reduced the levels of serum TNF-alpha induced by an intravenous pulse of LPS. The serum of pretreated mice blocked TNF-alpha production by peritoneal macrophages. The drug increased the levels of TNF-alpha regulators such as IL-10 and corticosteroids, whereas it inhibited TNF-alpha-dependent IL-6 production. Further TNF-alpha-dependent responses, such as cell extravasation, was decreased in treated mice. According to these results, Inmunoferon is postulated as an inhibitor of the systemic response to LPS. Correlation of the observations made in mice with a rat model suggests the efficacy of this product in reducing TNF-alpha production in a species-independent fashion and opens the possibility of its trial as an adjuvant of antibiotics in treatment against gram-negative bacterial infection.

Adjuvants, Immunologic↗

A novel class of carbonic anhydrase inhibitors: glycoconjugate benzene sulfonamides prepared by "click-tailing".

Aryl and heteroaryl sulfonamides (ArSO(2)NH(2)) are therapeutically used to inhibit the catalytic activity of carbonic anhydrases (CAs). Using a "click-tail" approach a novel class of glycoconjugate benzene sulfonamides have been synthesized that contain diverse carbohydrate-triazole tails. These compounds were assessed for their ability to inhibit three human CA isozymes in vitro: cytosolic hCA I and hCA II and transmembrane, tumor-associated hCA IX. This isozyme has a minimal expression in normal tissue but is overexpressed in hypoxic tumors and its inhibition is a current approach toward new cancer therapies. The qualitative structure-activity for all derivatives demonstrated that the stereochemical diversity present within the carbohydrate tails effectively interrogated the CA active site topology, to generate several inhibitors that were potent and selective toward hCA IX, an important outcome in the quest for potential cancer therapy applications based on CA inhibition.

Benzene Derivatives↗

Three-component Biginelli cyclocondensation reaction using C-glycosylated substrates. Preparation of a collection of dihydropyrimidinone glycoconjugates and the synthesis of C-glycosylated monastrol analogues.

The aldehyde-ketoester-urea cyclocondensation reaction has been revisited using C-glycosylated reagents with the aim of exploring a potential entry to a library of dihydropyrimidinone glycoconjugates. A collection of 13 mono- and bis-C-glycosylated dihydropyrimidinones has been prepared by a parallel synthesis approach using the three-component promoter CuCl/AcOH/BF(3) x Et(2)O. The sugar residues have been installed at either N1, C4, or C6 in the monoglycosylated derivatives and at both the C4 and C6 in the bisglycosylated products. The mono- and bisglycosylated products at C4 and C6 were obtained as mixtures of diastereoisomers with good to excellent selectivities due to the asymmetric induction by the sugar residue in the formation of the C4 stereocenter of the dihydropyrimidinone ring. Individual stereoisomers were isolated as pure compounds and their structures assigned with the aid of X-ray crystallography and chiroptical properties. As a demonstration of this new concept in the Biginelli reaction, the synthesis of two C4 epimer monastrol analogues bearing the ribofuranosyl moiety at C6 has been described.

Journal Article↗

"Click chemistry" inspired synthesis of pseudo-oligosaccharides and amino acid glycoconjugates.

[reaction: see text] Various pseudo-oligosacchardies and amino acid glycoconjugates were synthesized via an intermolecular 1,3-dipolar cycloaddition ("click") reaction using easily accessible carbohydrate and amino acid derived azides and alkynes as building blocks. It is pertinent to mention that the conjugation reaction is highly regioselective and high yielding and can be carried out under mild reaction conditions.

Alkynes↗

Synthesis of Glycoconjugate Vaccines against Shigella dysenteriae Type 1.

Syntheses of a hexadecasaccharide and smaller fragments corresponding to one-four repeating units of the O-specific polysaccharide of Shigella dysenteriae type 1 are reported in a reactive aglycon-linked from. Two tetrasaccharide donor/acceptor repeating units were assembled from monosaccharide precursors in a stepwise fashion and used in a linear, iterative manner to construct the higher-membered saccharides using Schmidt's glycosylation technique that proved superior to others tested. Single-point attachment of the saccharides to human serum albumin, using a secondary heterobifunctional spacer, afforded a range of glycoconjugates for a detailed evaluation of their immunological characteristics.

Journal Article↗

Synthesis of chitooligomer-based glycoconjugates and their binding to the rat natural killer cell activation receptor NKR-P1.

NKR-P1 protein is an important activating receptor at the surface of the rat natural killer cells. GlcNAc and chitooligomers were identified as strong activation ligands in vitro and in vivo. Their clustering brings about increase of their affinity to the NKR-P1 by 3-6 orders. Here we describe novel methodology for preparation of neoglycoproteins based on BSA carrying the chitooligomers (n = 2-5). Further on we developed novel methodology of the coupling of glycosylamines via aromatic-SCN activated linker both to protein or synthetic cores. Inhibition studies of chitooligomer glycoconjugates with the NKR-P1 receptor show that our neoglycoproteins are very strong ligands with high binding affinity (-log IC(50) = 13-15). In analogy with our previous observations with GlcNAc clustered on protein or PAMAM backbones the synthetic chitooligomer clusters should provide considerably better ligands in the in vivo antitumor treatment.

Amines↗

Incorporation of l-[3H]fucose and d-[3H]glucosamine into cell-surface-associated glycoconjugates in epidermis of cultured pig skin slices.

1. Electron microscope autoradiography indicated that L-[3H]fucose and D-[3H]glucosamine were both incorporated into cell-surface-associated glycoconjugates in the epidermis of cultured pig skin slices. 2. Acid hydrolysis and paper chromatography of skin homogenates confirmed that there was little metabolic conversion of the labeled precursors to other sugars. 3. Epidermis was separated from dermis using CaCl2, and was extracted with 8 M-urea/5% (w/v) sodium dodecyl sulphate and was then analysed by gel electrophoresis. The major component labelled with D-[3H]glucosamine had an apparent molecular weight in excess of 200 000. This material was not labelled with L-[3H]fucose. Lower molecular-weight components were labelled to a similar extent with both L-[3H]fucose and D-[3H]glucosamine. 4. The high molecular-weight material labelled with D-[3H]glucosamine was released into the medium when the epidermal cells were dispersed with trypsin, indicating that it was either surface-associated or was extracellular. It was also labelled with D-[14C]glucuronic acid, 35SO4(2-) and to a small extent with 14C-labelled amino acids indicating that it contained glycosaminoglycans derived from epidermal proteoglycans. This was confirmed by the fact that it was degraded by testicular hyaluronoglucosidase. It was not present in isolated membranes but was recovered in the soluble fraction from epidermal homogenates. It is therefore only very loosely bound at the cell surface or is present in the extracellular spaces. 5. Membrane-bound [3H]glycoproteins were identified after differential centrifugation of epidermal homogenates. The radioactivity profiles of membrane glycoproteins were similar whether L-[3H]fucose or D-[3H]glucosamine were used and both consisted of a major heterogeneous peak in the apparent mol.wt. range 70 000--150 000. [3H]Glycoproteins in this molecular-weight range were also major components of a plasma-membrane-enriched fraction. These glycoproteins were probably bound to the membrane by hydrophobic interactions, since they were only solubilized by treatment with detergent or organic solvent. They contained terminal sialic acid residues, since they were degraded by neuraminidase.

Animals↗

The N-acetylgalactosamine residue of exogenous GM2 ganglioside is recycled for glycoconjugate biosynthesis in rat liver.

A metabolic recycling of N-acetylgalactosamine (GalNAc), liberated from exogenous GM2 ganglioside [nomenclature of Svennerholm (1964) J. Lipid Res. 5, 145-155; IUPAC-IUB recommendations (1977) Lipids 12, 455-468], is demonstrated in rat liver. After the injection of a GM2 ganglioside isotopically radiolabelled on the terminal GalNAc residue ([GalNAc-3H]GM2), the liver retained a large amount of radioactivity distributed among: (1) a glycoprotein/glycosaminoglycan fraction, (2) a ganglioside fraction; and (3) a free-sugar fraction. Furthermore, volatile radioactivity was also found. The relative incorporation in the above fractions was time-dependent. The glycoprotein/glycosaminoglycan fraction contained radioactivity that was located on the GalNAc and GlcNAc residues. The ganglioside fraction was composed of two main families: gangliosides formed by a recycling of the liberated GalNAc, and gangliosides derived by direct utilization of the administered GM2. The free-sugar fraction contained mainly GalNAc. We suggest that GalNAc, after being released in the course of intra-lysosomal ganglioside catabolism, crosses the lysosomal membrane and passes into the cytosol, where the part not degraded is re-utilized for the biosynthesis of the different glycoconjugate classes.

Acetylgalactosamine↗

Primary structure of a 120 kDa protein associated with the fucose sulfate glycoconjugate constituting the acrosome reaction-inducing substance of the sea urchin, Hemicentrotus pulcherrimus.

A fucose sulfate glycoconjugate (FSG), a natural acrosome reaction-inducer, was purified from the egg jelly of the sea urchin Hemicentrotus pulcherrimus. The FSG is composed primarily of four constituents: a 120 kDa protein, a 237 kDa protein, a 258 kDa protein, and a polysaccharide-containing protein. Among them, the 120 kDa protein was thought to play a critical role in the association of other FSG constituent proteins, and therefore was characterized from a structural point of view. The protein was isolated from the carboxymethylated FSG by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and then digested with trypsin to obtain information regarding the primary structure. Based on the partial amino acid sequences of three internal peptides (FSG120KA: LHNNEYGYGDTAAGEPELAQEEID, FSG 120KG: AIDIPAETGHYGR, and FSG120KC: RPTFDLADAVDT) and the N-terminal peptide (LHNNEYGYGDTAAGEPELAQQEID) of the 120 kDa protein obtained from intact FSG, degenerate oligonucleotide primers were synthesized and used to amplify a 297 bp cDNA fragment. This fragment enabled us to obtain the full-length cDNA (3176 bp) by 5'- and 3'-rapid amplification of cDNA ends. The deduced amino acid sequence revealed that the 120 kDa protein is composed of 663 amino acid residues including 72 cysteine residues, and hence, about 40% is presumed to be carbohydrate by weight. The 120 kDa protein plays an important role in the association of FSG constituent proteins (258 and 237 kDa) through disulfide bonds.

Acrosome Reaction↗

Evidence for receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages.

Alveolar macrophages have been shown to bind glycoproteins and synthetic glycoconjugates (neoglycorpoteins) that have mannose, N-acetylglucosamine, or glucose in the exposed, nonreducing position. Galactose-terminal glycoproteins are not bound. Binding of radiolabeled ligands to cells is nearly completely impaired by the presence of an excess of yeast mannan. Binding is temperature sensitive and proceeds optimally at pH 7.0. Prior treatment of the cells with trypsin severely decreases their capacity to bind ligands. An inhibition assay has been developed, using radioiodinated glucose-albumin conjugate, agalacto-orosomucoid, beta-glucuronidase, and RNase B as ligands. Various glycoproteins have been shown to be effective inhibitors of ligand binding including horseradish peroxidase, agalacto-orosomucoid, beta-glucuronidase, ovalbumin, agalacto-fetuin, and RNase B. RNase A and asialo-fetuin are ineffective as antagonists. The results suggest the presence of a cell surface receptor on alveolar macrophages that binds glycoproteins having terminal sugars with the mannose or glucose configuration.

Animals↗

Stability of carbohydrate-modified vesicles in vivo: comparative effects of ceramide and cholesterol glycoconjugates.

The stability and tissue distribution of lipid vesicles modified at the surface by the incorporation of either a galactosyl ceramide (GalCer) or a galactosyl cholesterol (GalChol) glycoconjugate have been studied in mice by measuring the release of vesicle-entrapped 111In. Although the tissue distributions of both vesicle types were similar, the GalCer-containing vesicles were markedly less stable than those prepared with GalChol, whether administered orally or by intraperitoneal injection. Physical characterization of the vesicles in vitro suggests that the increased disruption rate for GalCer vesicles in vivo is related to structural instabilities induced by the cerebroside, which can then result in either an increased rate of vesicle uptake by tissues or a greater susceptibility to lysis. These studies demonstrate the importance of the nonpolar anchoring groups in determining the fate of surface-modified vesicles in vivo.

Administration, Oral↗