The effects of monensin on blood-borne arrest and glycosylation of BL/VL3 lymphoma cells.

We have previously shown that inhibitors of N-glycan processing alter both the cell surface carbohydrates and the homing properties in lymphoid cells. We have now studied the effects of the ionophore monensin (MON) on these parameters. Arrest in the spleen of [111In]-labelled BL/VL3 murine T lymphoma cells, injected intravenously was clearly reduced if the cells had been cultured for 24 h in the presence of monensin (0.1-1.0 microgram ml-1). We have characterized glycopeptides from BL/VL3 murine T lymphoma cells. Following labelling with tritiated precursors (fucose, mannose, galactose, glucosamine), surface glycopeptides from BL/VL3 murine T lymphoma cells, were released by trypsin and separated by gel filtration on Bio-Gel P6 and by affinity chromatography on immobilized lectins. After treatment with MON, a class of high molecular mass glycopeptides was no longer found. There were less complex and more high mannose glycans, as a consequence of a reduction of terminal glycosylation (sialylation, fucosylation or incorporation of N-acetyl-glucosamine). Similar findings were obtained with immunoprecipitated Thy-1 antigen. However, as estimated by flow cytometry analysis, the cell surface expression of Thy-1 was not reduced in MON-treated cells. Taken together our results show that cell surface oligosaccharides are modified dramatically, but that at least, certain cell surface antigens are present in normal amounts. It is tempting to speculate that changes in glycosylation account for the abnormal homing properties of MON-treated cells.     

Modification of oligosaccharide-lipid synthesis and protein glycosylation in glucose-deprived cells

Incubation of vesicular stomatitis virus-infected glucose-starved baby hamster kidney cells with [³⁵S]methionine results in the synthesis of all viral proteins. However, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping, the G protein is abnormally glycosylated. Metabolic labeling of the oligosaccharide-lipid precursors with [³H]mannose for 15 min, followed by Chromatographic and enzymatic analysis, indicates that the radiolabeled lipid-linked oligosaccharides are devoid of glucose in contrast to the glucosylated oligosaccharide-lipids synthesized by cells grown in the presence of glucose. Also, in contrast to control cells, examination of the glycopeptide fraction reveals the presence of [³H]mannose-labeled glycopeptides which are resistant to erado-β-N-acetylglucos-aminidase H and are smaller in size than glycopeptides from mature vesicular stomatitis virus. In order to observe these effects, a minimum time of 5 h of glucose deprivation is necessary and the addition of 55 μm glucose or mannose to the medium reverses these effects. These results indicate that vesicular stomatitis virus-infected BHK cells deprived of glucose are unable to glucosylate the oligosaccharide-lipid intermediates and, consequently, are unable to glycosylate the G protein normally.     

The role of glycoconjugates in metastatic melanoma blood-borne arrest and cell surface properties

The role of glycoconjugates in cell surface and blood-borne implantation properties of murine metastatic melanoma sublines of low (B16-F1) or high (B 16-F10) potential to colonize lungs was investigated by treating melanoma cells with the antibiotic tunicamycin. This drug prevents glycosylation of glycoproteins by inhibiting the formation of lipid-linked oligosaccharide precursors. The degree of tunicamycin-mediated modifications in glycoproteins was assessed by monitoring the decrease in cell surface sialogalactoproteins by binding of ¹²⁵I-labeled Ricinus communis agglutinin I. Scanning electron microscopy of tunicamycin-treated B16-F1 and B16-F10 cells showed morphologic changes such as cell rounding and formation of numerous surface blebs. Tunicamycin-treated B16-F1 and B16-F10 cells lost their lung colonization abilities when injected intravenously into C57BL/6 mice, concomitant with lowered rates of adhesion to endothelial cell monolayers, endothelial extracellular matrix (basal lamina), and polyvinyl-immobilized fibronectin in vitro, suggesting that this drug inhibits experimental metastasis by modifying the surface glycoproteins involved in determining the adhesive properties of malignant cells.     

Modification of the protein glycosylation pathway in the methylotrophic yeast Pichia pastoris

-1,2-Mannosidase from Trichoderma reesei was used to modify the N-linked glycosylation pathway of the methylotrophic yeast Pichia pastoris. Expression of foreign influenza glycoproteins with more extensively processed N-linked oligosaccharides was observed when -1,2-mannosidase was secreted in the culture medium. However, intracellular removal of mannose residues may stimulate mannosyltransferase activity and leads to hyperglycosylation. [3H]Mannose suicide selection or high concentrations of orthovanadate, commonly used to isolate glycosylation mutants of Saccharomyces cerevisiae, had no profound effect on Pichia pastoris. © Rapid Science Ltd. 1998     

Modification of a recombinant GPI-anchored metalloproteinase for secretion alters the protein glycosylation

The N-linked glycans of recombinant leishmanolysin (GP63) expressed as a glycosylphosphatidylinositol (GPI)-anchored membrane protein or modified for secretion in Chinese hamster ovary (CHO) cells were analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The glycans isolated from both membrane and secreted protein were predominantly complex biantennary structures. However other aspects of the glycan profiles showed striking differences. The degree of sialylation of the membrane form was greatly reduced and the core fucosylation of biantennary structures was increased compared to the secreted form. Glycans isolated from membrane expressed protein also contained a higher proportion of lactosamine repeats. Residence times in the secretory pathway were similar for both secreted and membrane protein. Glycosylation differences may therefore be due to differences in protein conformation and accessibility to glycosyltransferases or glycosidases. These differences in glycosylation represent an important factor when considering modifying membrane expressed proteins for secreted production.     

Modification of glycosylation reduces microvilli on rat liver epithelial cells

Effect of glycosylation modification on the shape of microvillus was investigated on rat liver epithelial cell line WB-F344. Since rat ER alpha-mannosidase has 89% identity to human 6A8 alpha-mannosidase, we used an antisense 6A8 cDNA fragment to inhibit expression in WB-F344 cells. Cells were transfected with antisense 6A8, the relevant sense fragment or the mock plasmid. Genomic PCR for neo(R) demonstrated integration of the transfected gene into host DNA. Enzymatic activity assay on p -nitro-phenyl-alpha-D-mannopyranoside showed suppression of ER alpha-mannosidase expression in the cells transfected with antisense 6A8. Concanavalin A binding to these cells was enhanced, indicating a modification in glycosylation. Number reduction and blunting of microvillus on these cells was observed. Transduction with the sense fragment or the mock had no effect. Cells with suppressed ER alpha-mannosidase expression grew slower in culture. Our results indicate an obvious effect of glycosylation modification on microvilli, which might be related with malfunctioning of cells.     

毕赤酵母蛋白质糖基化途径的改造

【背景】以酵母为宿主生产的蛋白往往发生过糖基化,形成高甘露糖型的N-糖基化。高甘露糖型的结构易在人体中引起免疫反应,这是酵母不能用于绝大部分糖蛋白药物生产的主要限制因素。因此,构建表达人源糖基化糖蛋白的酵母底盘细胞将为糖蛋白药物的生产提供强有力的工具。库德里阿兹威氏毕赤酵母(Pichia kudriavzevii)具有极强的抗逆性且生长迅速,是一种近年来备受关注的非典型性酵母,对其进行糖基化途径的改造将具有巨大的应用前景。【目的】对酵母N-糖基化途径的改造,首先要使其N-糖基化转变为Man₅GlcNAc₂核心结构,本研究对P. kudriavzevii的och1基因进行敲除并引入源自曲霉的msd S基因,以改变其分泌糖蛋白N-糖链的糖型结构。【方法】通过基因编辑对P. kudriavzevii的N-糖基化途径进行改造,获得P. kudriavzeviiΔura3Δoch1::msd S菌株,分析P. kudriavzeviiΔura3Δoch1::msd S菌株分泌糖蛋白上N-糖组的变化。【结果】与野生型P. kudriavzevii相...     

Modification of redox properties of cells by ortho-benzoquinones

Ortho-benzoquinones have been studied for their effect on the processes of methemoglobin formation in the rabbit erythrocytes and reduction of potassium ferricyanide by the HeLa cells. It is established that the incubation of cells in the presence of lipophilic quinone OBQ-1 results in the formation of the intracellular methemoglobin in erythrocytes and the intensification of the ferricyanide reduction by HeLa cells. OBQ-2 differing in the presence of the polar sulphogroup does not react with the intracellular oxyhemoglobin and exerts no effect on the ferricyanide reduction. It is supposed that OBQ-1 may change the ratio of the oxidized and reduced metabolites in a cell, thus inducing in it a state of the oxidative stress. A conclusion is drawn hat ortho-benzoquinones are able to modify the redox properties of cells, the efficiency of modification depending on the chemical structure of the compounds.     

Influence of glycosylation inhibitors on dihydropyridine binding to cardiac cells

In primary cultures of neonatal rat heart cells we found a linear correlation between the number of L-type calcium channel-specific dihydropyridine (DHP) binding sites and spontaneous beating frequency (v).Formation of glycoproteins in tissue culture was suppressed by different inhibitors of N-glycosylation. This inhibition alters to a different extent the binding of the DHP ligand (+)-[methyl-³H]PN 200-110 and v. The most severe but reversible effect occurs at 6 g/ml tunicamycin (B_max 45% and v 6%, resp., of control), a slight increase in B_max at 0.1–0.5 mM castanospermine and 0.05–2.5 mM deoxymannojirimycin. The other inhibitors gave no significant alterations of B_max.     

Synergism of endogenous inhibitors, exogenous cytostatic agents and hormones in arrest of proliferation

The synergism between endogenous regulators of proliferation (protors), alkylating agents and hormones in vitro was studied. The effects were monitored by the incorporation of 3H-TdR into human and rat short term bone marrow cultures and by the formation of mouse granulocyte-macrophage colonies in semisolid agar capillaries. An additive and/or slight potentiating synergism was demonstrated between different types of inhibitory protors (GI-3S2, GI-3S3 and GI-3B), between GI-3 and hydrocortisone, and between GI-3 and the alkylating agents (adriamycin, dianhydrogalactitol) examined. The results offer a real possibility of strengthening the inhibition of neoplastic proliferation without increasing cytotoxicity of the drugs used.     

Effect of endogenous lectins on HCO(3)-ATPase activity of the brain glia cells

The influence of galactose--(GL-GAL) and inositol-specific (GL-I) lectins from the glial cells of the chicken brain fraction on the HCO3- -ATPase activity was studied. It was established that enzyme activity changes depended on the concentration of lectins. It must be said that the presence of these lectins also changes the pH optimum of enzyme activity. Calcium ions have an inhibitory effect on the HCO3- -ATPase activity. This effect sharply decreases as a result of the presence of GL-GAL and GL-I. The modulator influence of lectin on the HCO3- -ATPase activity is determined by changing the enzyme affinity for Ca2+ ions.     

Investigating the glycosylation of normal and ovarian cancer haptoglobins using digoxigenin-labelled lectins

Human haptoglobin (Hp), prepared from 10 normal sera and 10 ovarian cancer sera as well as from 11 ovarian cancer ascitic fluids, was characterized with regard to its reactivities with different lectins. Digoxigenin-labelled lectins [peanut agglutinin (PNA),Arachis hypogaea; SNA,Sambucus nigra; MAA,Maackia amurensis; DSA,Datura stramonium; and Con A, concanavalin A] with different carbohydrate specific moieties were used to identify sugar structures in Hp by blotting and by a quantitative assay in multiwell plates [lectin/enzyme-linked immunosorbent assay (ELISA)]. It was found that the lectin blotting was only useful for preliminary investigations, but that the lectin/ELISA gave interesting results that indicated the presence ofN-linked complex chains. Despite the fact that PNA interacted weakly with desialylated Hp in lectin blotting, no other evidence was obtained to suggest the presence ofO-linked glycans. Quantitative differences between normal and cancer Hp were observed for Con A, SNA and MAA, but no difference was found in the reaction with DSA. The binding of cancer Hp to Con A and SNA was twice that of normal Hp. Normal serum and ascitic fluid Hp bound similar amounts of MAA, but three times that observed for cancer serum Hp. Our results suggest that normal and ovarian cancer Hp differ in the content of carbohydrate structures containing sialic acid linked (2–6) or (2–3) to galactose and the type of oligosaccharide branching.     

Hepten inhibitors of the endogenous galactose binding lectins and anti-lectin antibodies inhibit primitive streak formation in the early chick embryo

The early chick blastoderm expresses two endogenous galactose-bindinglectins of 14 kDa and 16 kDa. We have studied the effect thelectin hapten inhibitors thiodigalactoside and the syntheticneoglycoprotein lactosyl-bovine serum albumin as well as polyclonalanti-lectin antibodies on the development of early chick embryoscultured in a defined medium. Controls consisted of maltose,maltosyl bovine serum albumin and rabbit IgG. Embryos treatedat the onset of cell migration during early gastrulation underwentblastoderm retraction with decrease in surface area. In addition,they exhibited a lack of demarcation between the presumptiveembryonic area (area pellucida) and the presumptive extraembryonicarea (area opaca). These blastoderms also lacked a primitivestreak, that is, the structure that forms in the area pellucidaduring gastrulation as cell migrate to form the endodermal andmesodermal layers of the embryo. Embryos treated at later stagesof gastrulation showed development similar to that of controlsin that they were able to undergo early organogenesis. The resultssuggest that lectin mediated mechanisms are essential for themigratory movements of early gastrulation and that, at lategastrulation, other mechanisms exist in the embryo to compensatefor lectin function. blastoderm chick embryo galectin     

Inhibitors of protein glycosylation inhibit the differentiation of Friend erythroleukemia cells

Tunicamycin, 2-deoxy-d-glucose and 2-deoxy-2-fluoro-d-glucose inhibit dimethyl sulfoxide-induced differentiation of Friend cells. This inhibition, characterized by inhibition of hemoglobin synthesis, is accompanied by a specific inhibition of protein glycosylation. The results of cloning experiments indicate that this inhibition specifically affects cells in the period preceding their commitment. These results suggest that glycoprotein synthesis is a requirement for Friend erythroleukemia cells in order to initiate the expression of the terminal differentiation program.     

Analysis of protein glycosylation by mass spectrometry

There is a growing pharmaceutical market for protein-based drugs for use in therapy and diagnosis. The rapid developments in molecular and cell biology have resulted in production of expression systems for manufacturing of recombinant proteins and monoclonal antibodies. These proteins are glycosylated when expressed in cell systems with glycosylation ability. For glycoproteins intended for therapeutic administration it is important to have knowledge about the structure of the carbohydrate side chains to avoid cell systems that produce structures, which in humans can cause undesired reactions, e.g., immunological and unfavorable serum clearance rate. Structural analysis of glycoprotein oligosaccharides requires sophisticated instruments like mass spectrometers and nuclear magnetic resonance spectrometers. However, before the structural analysis can be conducted, the carbohydrate chains have to be released from the protein and purified to homogeneity, and this is often the most time-consuming step. Mass spectrometry has played and still plays an important role in analysis of protein glycosylation. The superior sensitivity compared to other spectroscopic methods is its main asset. Structural analysis of carbohydrates faces several problems, however, due to the chemical nature of the constituent monosaccharide residues. For oligosaccharides or glycoconjugates, the structural information from mass spectrometry is essentially limited to monosaccharide sequence, molecular weight, and only in exceptional cases glycosidic linkage positions can be obtained. In order to completely establish an oligosaccharide structure, several other structural parameters have to be determined, e.g., linkage positions, anomeric configuration and identification of the monosaccharide building blocks. One way to address some of these problems is to work on chemical pretreatment of the glycoconjugate, to specifically modify the carbohydrate chain. In order to introduce specific modifications, we have used periodate oxidation and trifluoroacetolysis with the objective of determining glycosidic linkage positions by mass spectrometry.     

Analysis of protein glycosylation by mass spectrometry

We present a detailed protocol for the structural analysis of protein-linked glycans. In this approach, appropriate for glycomics studies, N-linked glycans are released using peptide N-glycosidase F and O-linked glycans are released by reductive alkaline beta-elimination. Using strategies based on mass spectrometry (matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano-electrospray ionization mass spectrometry/mass spectrometry (nano-ESI-MS-MS)), chemical derivatization, sequential exoglycosidase digestions and linkage analysis, the structures of the N- and/or O-glycans are defined. This approach can be used to study the glycosylation of isolated complex glycoproteins or of numerous glycoproteins encountered in a complex biological medium (cells, tissues and physiological fluids).     

Sequence conservation in the chagasin family suggests a common trend in cysteine proteinase binding by unrelated protein inhibitors

The recently described inhibitor of cysteine proteinases from Trypanosoma cruzi, chagasin, was found to have close homologs in several eukaryotes, bacteria and archaea, the first protein inhibitors of cysteine proteases in prokaryotes. These previously uncharacterized 110-130 residue-long proteins share a well-conserved sequence motif that corresponds to two adjacent beta-strands and the short loop connecting them. Chagasin-like proteins also have other conserved, mostly aromatic, residues, and share the same predicted secondary structure. These proteins adopt an all-beta fold with eight predicted beta-strands of the immunoglobulin type. The phylogenetic distribution of the chagasins generally correlates with the presence of papain-like cysteine proteases. Previous studies have uncovered similar trends in cysteine proteinase binding by two unrelated inhibitors, stefin and p41, that belong to the cystatin and thyroglobulin families, respectively. A hypothetical model of chagasin-cruzipain interaction suggests that chagasin may dock to the cruzipain active site in a similar manner with the conserved NPTTG motif of chagasin forming a loop that is similar to the wedge structures formed at the active sites of papain and cathepsin L by stefin and p41.