Carbohydrate Moieties Of Glycoproteins Research Articles

The Role of Lectin as Potential Biomarker in Ovarian Cancer.

Lectin acts as an effective tool for screening potential biomarkers and gives an indication of highly valued research. Lectin offers the advantage of having the ability to recognize carbohydrate moiety of glycoprotein, peptidoglycan, glycosides, glycopeptides, lipopolysaccharide etc. makes powerful aid in detecting new cancer biomarkers in most complex tissues and fluids. The unique specificity of lectin in detecting single out anomalously expressed glycosylation pave lectin-based method that can often go down the line for future cancer biomarkers. This article explores the different types of lectin, their sources and possible application in masking the activity of ovarian cancer cells.

Roles and Biomedical Applications of Haemolymph Lectin.

Lectins are class of proteins characterized by their ability to selectively bind carbohydrate moieties of glycoproteins. Many invertebrate lectins, especially derived from hemolymph, are being purified, and yet their functions and medical applications are subjects of major interest. Hemolymph lectins in invertebrates play a major role in protecting against many pathogens and microbes. Further, many hemolymph lectins show anticancer properties towards various cancer cell lines, which expresses globotriaosyl ceramides on their cell surface. These vast repertoires of hemolymph lectins in recognizing and inhibiting the growth of various harmful microbes and cancerous cells have spurred the biochemist to use them in histochemical and cytochemical studies. The present review will address the biological roles and biomedical applications of hemolymph lectin.

β-1,4-Galactosyltransferase-involved in lipopolysaccharide-induced adhesion of schwann cells

The carbohydrate moieties of glycoprotein are associated with some inflammatory diseases by affecting a wide range of biological functions of cells. This study aimed to investigate the role of β-1,4-galactosyltransferase-I (β-1,4-GalT-I) in adhesion of Schwann cells during inflammation. A rat Schwann cell line, RSC 96 was used. We used western blotting to detect the expression of β-1,4-GalT-I. Flow cytomety was used to measure the galactosylation of glycoproteins on cell surfaces. Immunofluorescent staining was used to examine the expression of α6 integrin, focal adhesion kinase (FAK) and F-actin. Tyrosine phosphorylation of FAK was detected by immunoprecipitation. An adhesion assay was performed to investigate the adhesion of Schwann cells. One-way ANOVA was used to compare differences between the operated and the control group. Schwann cell adhesion was induced by LPS stimulation and was accompanied by upregulation of β-1,4-GalT-I expression and galactosylation of glycoproteins. There was a change of localization of FAK and cytoskeleton organization in LPS treated cells compared with control cells. The pretreated cells enhanced tyrosine phosphorylation of FAK compared with control cells in the adhesion process. With the increased cell surface expression of α6 integrin and β-1,4-GalT-I, the adhesion of Schwann cells on laminin was increased as well. These results suggested that β-1,4-GalT-I may play an important role in adhesion of Schwann cells during inflammation.

Structural Analyses of Carbohydrate Moieties of Glycoproteins by Microwave-Assisted Partial Acid Hydrolysis and Mass Spectrometry

Characterization of carbohydrate moieties of glycoproteins using microwave-assisted partial acid hydrolysis (MAPAH) and mass spectrometry (MS) are described in this review. Acids including hydrochloric acid (HCl), trifluoroacetic acid (TFA), and phosphoric acid (H3PO4) can be used to induce partial hydrolysis of the carbohydrate moieties of glycoproteins in as short as 30 s of microwave exposure. High resolving power of MS allows distinction between different glycopeptide fragments. Several glycoproteins including bovine pancreatic ribonuclease B (RNase B, MW ∼15 kDa), egg white avidin (MW ∼16 kDa), human serum α1-acid glycoprotein (α1-AGP, MW ∼36 kDa), horseradish peroxidase (HRP, MW ∼44 kDa), fetal calf serum fetuin (BSF, MW ∼45 kDa), and glucose oxidase from Aspergillus niger (GO, MW ∼75 kDa), are used to demonstrate this technique. Information of the sugar composition and/or arrangement of the carbohydrate moieties can be readily obtained either from glycoproteins directly or digested glycopeptides of the glycoproteins. This method is fast, sensitive, and a reliable technique. Challenges when working on larger and heavily glycosylated glycoproteins are discussed.

A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.

Adhesins of Enterotoxigenic Escherichia coli Strains That Infect Animals.

The first described adhesive antigen of Escherichia coli strains isolated from animals was the K88 antigen, expressed by strains from diarrheic pigs. The K88 antigen was visible by electron microscopy as a surface-exposed filament that was thin and flexible and had hemagglutinating properties. Many different fimbriae have been identified in animal enterotoxigenic E. coli (ETEC) and have been discussed in this article. The role of these fimbriae in the pathogenesis of ETEC has been best studied with K88, K99, 987P, and F41. Each fimbrial type carries at least one adhesive moiety that is specific for a certain host receptor, determining host species, age, and tissue specificities. ETEC are the most frequently diagnosed pathogens among neonatal and post-weaning piglets that die of diarrhea. Immune electron microscopy of animal ETEC fimbriae usually shows that the minor subunits are located at the fimbrial tips and at discrete sites along the fimbrial threads. Since fimbriae most frequently act like lectins by binding to the carbohydrate moieties of glycoproteins or glycolipids, fimbrial receptors have frequently been studied with red blood cells of various animal species. Identification and characterization of the binding moieties of ETEC fimbrial adhesins should be useful for the design of new prophylactic or therapeutic strategies. Some studies describing potential receptor or adhesin analogues that interfere with fimbria-mediated colonization have been described in the article.

Structural characterization of the nonameric assembly of an Archaeal α- l-fucosidase by synchrotron small angle X-ray scattering

α- l-Fucosidase is a lysosomal enzyme responsible for hydrolyzing the α-1,6-linked fucose joined to the reducing-end N-acetylglucosamine of carbohydrate moieties in glycoproteins. The first α- l-fucosidase from Archaea was recently identified in the genome of the hyperthermophile Sulfolobus solfataricus; the enzyme is encoded by two open reading frames separated by a −1 frameshift. A preliminary biochemical and biophysical characterization of this extremophile enzyme has been carried out both in solution, through small angle X-ray scattering experiments, and in the crystalline state, showing an unusual oligomeric assembly resulting from the association of nine subunits, endowed with 3-fold molecular symmetry.

The potential beneficial effect of glycine on the carbohydrate moieties of glycoproteins in an experimental model of alcohol-induced hepatotoxicity.

Glycine is known to have a protective role against alcohol-induced liver damage. The aim of our study was to evaluate the effect of glycine on liver and brain glycoproteins in alcohol-fed rats. Administering ethanol (7.9 g/kg of body of weight) every day to Wistar rats for 60 days resulted in significantly elevated levels of liver and brain hexosamine, fucose, and sialic acid and significantly reduced levels of total hexoses as compared with those of the control rats. Simultaneous glycine supplementation (0.6 g/kg of body weight) during the last 30 days of the experiment to rats given alcohol normalized the levels of hexosamine, fucose, and sialic acid and elevated the levels of total hexoses in the liver and brain significantly as compared with unsupplemented alcohol-treated rats. Microscopic examination of alcohol-fed rat liver showed inflammatory cell infiltrates and fatty changes, which were reversed on treatment with glycine. Similarly, alcohol-treated rat brain demonstrated edema, which was markedly reduced on treatment with glycine. Thus glycine administration plays a significant role in reducing the toxicity of ethanol.

Effect of Embelin on Carbohydrate Moieties of Glycoprotein in Tumour-Bearing Rats

Objective: To investigate the changes taking place in the carbohydrate moieties of the glycoprotein in plasma, liver and kidney of the tumour-bearing rats on embelin treatment. Materials and methods: Fibrosarcoma was induced to three groups of animals by injecting the tumour cell line. To two groups, embelin was administered after the tumour became palpable, in two doses (50 and 100 mg/kg) continuously, for 20 days. On 21st day, the animals were sacrificed, blood collected and liver and kidney separated. Total hexose, hexosamine and sialic acid in plasma, liver and kidney tissues were estimated. Results: In tumour-bearing control animals, the above parameters in plasma, liver and kidney tissues were elevated. Continuous administration of embelin lowered these values to near normal. Conclusion: Embelin significantly reversed the elevated levels of carbohydrate moieties of glycoprotein in tumour-bearing rats. This further supports the anti-tumour property of embelin.

Imparting bone affinity to glycoproteins through the conjugation of bisphosphonates.

To develop a novel means of conjugating bisphosphonates onto the carbohydrate moieties of glycoproteins to enhance protein affinity to bone. 1-Amino-1,1-diphosphonate methane (aminoBP) was conjugated onto the carbohydrate moietites of oxidized fetuin by using 4-(maleimidomethyl)cyclohexane-1-carboxyl-hydrazide (MMCCH). Bone affinity of the resulting conjugates was compared to proteins obtained from another means of conjugation, whereby aminoBP was conjugated onto fetuin's lysine moieties by using succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). The use of the MMCCH resulted in the conjugation of up to seven aminoBPs per molecule of fetuin. These conjugates gave a 2.6-, 2.0-, 30.5-. and 1.84-fold increased affinity for untreated, ashed, demineralized bone and hydroxyapatite, respectively, as compared to conjugates from the SMCC reaction. Both conjugates exhibited a pH-independent, equally slow degradation in adult bovine serum-containing media. The use of the MMCCH chemistry to conjugate aminoBP onto fetuin was feasible. Furthermore, the described processes of conjugation resulted in amino-BP-dependent increase in the glycoprotein's affinity to various bone matrices in a manner that exceeds the affinity produced by the previously established method, which used SMCC.

Effect of Curcumin on Carbohydrate Moieties of Glycoprotein in Alcohol-Induced Hepatotoxicity

ABSTRACT To obtain information about the changes in the carbohydrate moieties of glycoprotein in liver and the effect of curcumin on alcohol-induced toxicity, total hexose, fucose, and sialic acid were monitored. The activities of marker enzymes for hepatocellular damage, (alkaline phosphatase and aspartate transaminase) were significantly increased in rats fed alcohol as compared with control rats not given alcohol. A significant decrease in the level of total hexose and elevated levels of fucose and sialic acid was observed in rats fed alcohol as compared with controls. The administration of curcumin to rats fed alcohol decreased the activities of alkaline phosphatase and aspartate transaminase and the levels of fucose and sialic acid as compared with rats being given alcohol without curcumin, but increased the levels of total hexose.

ミルクオリゴ糖の糖修飾基情報から生理活性素材を創る！ 糖質化学分野における新マテリアルサイエンスの創成

It is well known that the carbohydrate moieties of glycoproteins or glycolipids, which exist on mammalian cell surfaces, are modified by sulfate or phosphate, etc, in a few cases. These modifiers are recognised as possible receptors in intercellular communications. On the other hand, mammalian milk or colostrum contains many kinds of free oligosaccharides other than lactose in common. These oligosaccharides are also suggested to have biological roles such as inhibition of the adhesion of pathogenic microorganisms to the intestinal tract of infant or brain development - stimulation in infants. Recently, a few milk oligosaccharides have also been shown to be modified and the biological significance of these saccharides are assumed as follows: 1) Sialyllactose lactone: Some proportion of Neu5Gc(α2-3)Gal(β1-4)Glc was found to occur as free lactones between the carboxyl group of Neu5Gc and OH of Gal or Neu5Gc itself (sialyllactose lactone) in ovine colostrum. The sialyl oligosaccharides in milk or colostrum may attach to bacterial toxin and viruses, such as influenza virus in the intestine, thus protecting the infant. The lactone can be assumed to be resistant to virus sialidase. When the sialic acid is liberated from sialyl oligosaccharides by virus sialidase, they would lose their protective effect but the lactone should retain this effect because of its resistance to the action of virus sialidase. 2) 4-O-acetylated sialyllactose: Neu5Ac(α2-3)Gal(β1-4) Glc, whose Neu5Ac was O -acetylated at OH-4, was identified in the milk of echidna, one of the monotremes. Although this saccharide was assumed to be a possible inhibitor for the adhesion of viruses or bacteria whose receptors are O -acetylated Neu5Ac, the exact function of this saccharide is still unknown. 3) sulfated oligosaccharides: Gal(β1-4)Glc-6'-O-sulphate (lactose 6'-O -sulfate) and Neu5Ac(α2-3)Gal(β1-4)Glc-6'-O-sulfate (N-acetylneuraminyllactose 6'-O-sulfate) were identified in rat milk, whereas Gal(β1-4)Glc-3'-O-sulfate (lactose 3'-O-sulfate) was found in dog milk. The presence of N-acetylneuraminyllactose 6'-O -sulfate is suggested in human milk, too, and, in addition, this milk contained oligosaccharides whose GlcNAc residues were replaced by sulfate at OH-6 position. These compounds may permit the simultaneous delivery of two essential nutrients, sulfate and calcium, in early life, avoiding the precipitation of insoluble calcium sulfate in milk. 4) phosphorylated oligosaccharides: Bovine or mare colostrum contained phosphorylated oligosaccha-rides including Neu5Ac(α2-6)Gal(β1-4)GlcNAc-a1-phosphate (cow), Neu5Ac(α2-6)Gal(β1-4)GlcNAc-6-phosphate (cow) and Gal(β1-4)GlcNAc-α1-phophate (horse). These oligosaccharides may also permit the simultaneous delivery of phosphate and calcium, avoiding the precipitation of calcium phosphate. The existence of these modified oligosaccharides in milk may offer valuable in-formation for manufacturing bio-functional materials in the food industry.

Quantitative analysis of oligosaccharide structure of glycoproteins

A sensitive and quantitative method for the structural analysis of oligosaccharide was established for the glycoform analysis of glycoproteins. In this study,N-linked oligosaccharides of human IgG and bovine transferrin were analyzed for the evaluation of the method. Carbohydrate moiety of glycoprotein was released by hydrazinolysis and purified by paper chromatography. The oligosaccharides were labeled with a fluorescent dye, 2-aminobenzamide, for the enhancement of detection sensitivity. Sialylated (acidic) oligosaccharides were separated from neutral oligosaccharide by employing a strong anion-exchange column (MonoO) followed by the treatment with sialidase. Enzymatically desialyated fractions and neutral fractions of oligosaccharides were applied to normal-phase HPLC to resolve the peaks according to glucose unit (GU). The structure of separated molecules was further determined by sequential digestion with exoglycosidases. As a result, disialylated biantennary complextype oligo saccharide was found to be a major sugar chain in bovine transferrin (63%). In human IgG, core fucosylated asialobiantennary complex oligosaccharides were dominant. These results coincided well with reported results.

Essential roles of carbohydrate signals in development, immune response and tissue functions, as revealed by gene targeting.

Knockout mice lacking glycosyltransferases or sulfotransferases provide unequivocal evidence that the carbohydrate moieties of glycoproteins, glycolipids, and proteoglycans play essential roles in various biological phenomena such as development, the immune response, and tissue functions. Examples of abnormalities of null mutants include arrest of embryogenesis due to deletion of N-acetylglucosaminyltransferase I or glucosylceramide synthase, failure of kidney formation in heparan sulfate 2-O-sulfotransferase deficiency, suppressed antibody production in alpha-2, 6-sialyltransferase deficiency, male sterility in GM2/GD2 synthase deficiency, and abnormalities in the function and stability of myelin in galactosylceramide deficiency.

Artificially glycosylated α-chymotrypsin in reversed micelles of Aerosol OT in octane: A new approach to elucidation of the role of carbohydrate moieties in glycoproteins

A comparative study of native and artificially glycosylated α-chymotrypsin in reversed micelles of Aerosol OT in octane was carried out. d-Glucosamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were used as modifying agents to yield glycosylated enzyme. Unlike the native α-chymotrypsin, the modified protein tended to form reversible oligomeric structures, revealed by the appearance of an additional maximum (characteristic of dimeric forms of protein functioning) as a result of the enzyme catalytic activity being dependent on the AOT hydration degree. Dependence of the enzyme catalytic activity on the surfactant concentration in the case of the modified enzyme was similar to that of glycoproteins, and suggests its membrane affinity. The role of carbohydrate moieties in the functioning of glycoproteins is discussed.

S7.10 Influence of amino sugar analogues on the activation of lymphocytes

The carbohydrate moieties in glycoproteins influence uptake and distribution and provide protection against enzymatic proteolysis. To investigate the influence of glycosylation on the conformation, metabolic stability, and pharmacological properties of peptides we have prepared the derivatives 1 4 of the antidiuretic hormone vasopressin. The glycopeptides 2 and 3 were prepared from N"-Fmoc-3O-(Ac4-/3-D-Gal)-Ser-OPfp by solid phase synthesis and purified by reversed phase HPLC (29 and 200/0 yields, respectively). Conformational studies using 1H-NMR spectroscopy showed that 1 3 populate similar conformations in H~O, which are independent of glycosylation and include a type II t-turn for the Cys 6 Gly 9 segment. We also found that glycosylation (compounds 2 and 3) abolished the antidiuretic effect of 1 and 4, but resulted in a significant increase in metabolic stability towards rat intestinal juice and in transport over the intestinal wall. Furthermore, in rats the doses of 2 and 3 available in plasma after oral administration increased 2 0 3 0 fold as compared to the drug DDAVP (1).

Bisected N-Acetylglucosamine Residue of Biantennary Sugar Chains and High-Molecular-Weight Oligosaccharides of Neuroblastoma Cell Membranes

The carbohydrate moieties of glycoproteins in plasma membrane are known to correlate with the induction of cell differentiation in some kinds of cells. We investigated the asparagine-linked sugar chains of neuroblastoma and ganglioneuroma cell membranes. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha-->(Man alpha)Man beta-->GlcNAc beta-->(+/- Fuc alpha-->)GlcNAc as their cores. A comparative study of the oligosaccharides of these cells showed that the biantennary complex-type sugar chain with bisecting N-acetylglucosamine residues increased and high-molecular-weight oligosaccharides decreased in ganglioneuroma cells. It is suspected that asparagine-linked sugar chains correlate with the differentiation stage of neuroblastoma.

Adhesins of escherichia coli

AbstractE. coli has got increasing importance as a causative agent of intestinal and extra‐intestinal diseases. In both these infections adhesion of the bacteria to mucous surface cells are initial events for coionization and development of infection. Adhesins are bacterial recognition proteins which specifically interact with carbohydrate moieties of glycoproteins or glycolipids on mammalian cells. The adhesiveness of bacteria is associated with filamentous surface appendages, designated as fimbriae or pili, as well as with non‐fimbrial components. Some recent data on the nomenclature, classification, disease association, receptor specificity, and topographic arrangement are presented. The correlation between E. coli O : K : H serovar and fimbrial antigens is demonstrated on the basis of E. coli isolated from patients with urinary tract infections. Hitherto unknown non‐fimbrial adhesins are briefly described.

Production of monoclonal antibodies directed against carbohydrate moieties of cell surface glycoproteins.

Through the use of a technique for raising monoclonal antibodies, coupled with a solid‐phase radioimmunoassay utilizing immobilized glyeopeptides prepared from the surface membranes of the colorectal cancer cells (LS 180) used for the immunization, carbohydrate‐directed monoclonal antibodies were obtained. One of the monoclonal antibodies, MLS 102, reacted immunohistochemically intensely with the colorectal cancer cell surface and the mutinous glycoproteins secreted by the cancer cells, but only weakly with normal colon tissue. The antigenic determinant recognized by MLS 102 was the carbohydrate moiety of glycoproteins with terminal sialic acid. The antigens defined by other monoclonal antibodies, MLS 103 and 104, were immunohistochemically detected in both normal colonic epithelial and cancer cells. These antibodies seemed to recognize the carbohydrate moieties of both glycoproteins and glycolipids. The method described in this report can be generally applied to raise cell surface carbohydrate‐directed antibodies.

A new endo-beta-galactosidase releasing Gal alpha 1—3Gal from carbohydrate moieties of glycoproteins and from a glycolipid.

Culture fluid of Clostridium perfringens contained an endoglycosidase releasing a galactosyl disaccharide from glycans derived from glycoproteins of teratocarcinoma OTT6050. The endoglycosidase was purified by ammonium sulfate precipitation, Sephadex G-200 column chromatography, and DEAE-Sephadex A-25 column chromatography. The structure of the disaccharide product was determined to be Gal alpha 1----3Gal. The enzyme also released the disaccharide from bovine thyroglobulin glycopeptide and from a pentaglycosyl ceramide (Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer). Therefore, we concluded that the enzyme was an endo-beta-galactosidase cleaving Gal beta 1----4GlcNAc linkage. Keratan sulfate and the antigenic determinant of blood group type B were resistant to the enzyme: the specificity of the enzyme is different from other endo-beta-galactosidases so far described. The enzyme could act on intact thyroglobulin and defucosylated ovarian cyst mucin.