Fluorous-tagged compound: a viable scaffold to prime oligosaccharide synthesis by cellular enzymes

Fluorous-tagged saccharide primers could be viable scaffolds for the synthesis of oligosaccharides. This research demonstrates that a fluorine-containing saccharide derivative could actually be taken up by the cell, the saccharide chain elongated by cellular enzymes, and the elongated product released by the cells to the culture medium. A fluorous-tagged lactoside primer, 6-(perfluorohexyl)hexyl-4-O-(beta-D-galactopyranosyl)-beta-D-glucopyranoside, was chemically synthesized and introduced in mouse B16 cells to prime oligosaccharide synthesis. Uptake of the primer by B16 cells resulted in the sialylation of the terminal galactose residue to afford an oligosaccharide with the same glycan structure as ganglioside GM3. The presence of many fluorine atoms did not have any adverse effects to the cells. Moreover, the number of fluorine atoms did not pose a steric barrier and instead, their presence possibly increased the hydrophobicity of the primer and enhanced membrane permeability. This strategy of using a fluorous-tagged primer and cells can pave the way for an easier way of preparing oligosaccharides via an environment-friendly approach that eliminates the use of large amounts of organic solvents.     

Further characterization of porcine plasma fibronectin which contains fucosylated carbohydrate chains

Porcine plasma fibronectin and its functional four fragments produced by cathepsin B digestion were examined for biological, immunochemical and biochemical properties. Native fibronectin, 150-kDa and 130-kDa fragments exhibited similar cell attachment-promoting activity to each other. In an Ouchterlony double immunodiffusion system, these three polypeptides formed a precipitin line with anti-fibronectin antiserum, while the 50-kDA and 30-kDa fragments did not. The 150-kDa and 130-kDa fragments contained free sulfhydryl(s). The glycopeptide fractions were prepared by pronase digestion of porcine and human plasma fibronectin, and radiolabeled with [¹⁴C]acetic anhydride. The results of affinity chromatography with concanavalin A and lentil lectin immobilized on agarose indicated that the porcine glycopeptide fraction was different from the human fraction in that a larger part (58%) of the former was bound to lentil lectin. About 90% of this lentil lectin-reactive glycopeptides lost this reactivity upon α-L-fucosidase digestion. The glycopeptide fractions were also prepared from three carbohydrate-containing domains. Less than 30% of the radioactivity of the glycopeptide fractions of 150-kDa and 130-kDa fragments was retained on the lentil lectin-agarose, while about 90% of that from the 50-kDa fragment was retained. These results indicate that porcine plasma fibronectin has characteristics very similar to those of human plasma fibronectin and others, but is unique in that it contains fucosylated carbohydrate chains which unevenly distribute through functional domains.     

An oligosaccharide component in proteoglycans of articular cartilage

Two types of sialic acid-containing component are released from articular cartilage proteoglycan monomer (D1) treated with 0.05 M NaOH containing 1 M NaBH₄. The smaller component, which has not been described before, contains galactosamine, glucosamine, galactose and sialic acid (Molar ratio 1:1:1:2). It is eluted from ECTEOLA-cellulose with low molarity (0.4 M) sodium formate and has a K_av of 0.70 on Bio-gel P30. Its presence on the proteoglycan monomer was demonstrated at all stages of foetal and adult life.     

Ultrastructural localization of fibronectin in bone marrow of the embryonic chick and its relationship to granulopoiesis

Summary Fibronectin was immunolocated in embryonic chick bone marrow by the use of both a direct peroxidase conjugated antiserum and an indirect Streptavidin bridge technique. Fibronectin is located in the extravascular granulopoietic compartment and, to a lesser extent, in the vascular, erythropoietic compartment. There is no evidence of fibronectin being associated with blood-stromal cell interactions involving either erythropoiesis or thrombopoiesis. However, mature thrombocytes display a substantial surface coat containing fibronectin. Much of the fibronectin appears to be situated on surfaces of those fibroblastic stromal cells which support granulopoiesis. Fibronectin containing extracellular material connects surfaces of developing granulocytes with surfaces of stromal cells. Fibronectin is a surface component of granulocytes as well as nearby stromal cells. However, there appear to be fewer ferritin particles per unit of surface on granulocytic cells. Many of the ferritin particles are not clearly associated with amorphous matrix material at cell surfaces. Immunocytochemical attempts to identify laminin were unsuccessful. These studies indicate that fibronectin is situated at sites where it could mediate adhesive interaction between granulopoietic cells and their stromal cells. Furthermore, cell surface-matrix interaction involving fibronectin could mediate migration of blood cells within the extravascular spaces.     

Characterization of oligosaccharide units of p-N-collagen type III from dermatosparactice bovine skin

p-N-collagen type III was extracted from dermatosparactic and normal fetal bovine skin and purified by ion-exchange chromatography using DEAE- and CM-cellulose. Asparagine-linked sugar chains were fractionated by high voltage paper chromatography from the products obtained after hydrazinolysis and reduction with NaB³H₄. These oligosaccharides composed of neutral and acidic components are mannose-containing oligosugars of the complex type. Their abundance is much higher in dermatosparactic p-N-collagen type III.     

Glycosylation and ligand-binding activities of rat plasma fibronectin during liver regeneration after partial hepatectomy

Fibronectin (FN) is a multifunctional glycoprotein present in the extracellular matrix (ECM) and plasma. We previously reported that the glycosylation and ligand-binding of vitronectin (VN) change markedly after partial hepatectomy (PH). Here we show the changes of FN during liver regeneration. The yields of purified sham-operated (SH-) and PH-FN were higher than that of non-operated (NO)-FN, while binding activities of FNs to ECM ligands were changed only slightly by hepatectomy. The carbohydrate concentration of PH-FN decreased to 66% of that of NO- and SH-FN. By using LC/MS(n), eight kinds of complex-type N-glycan structures were found to be present in all FNs, and bi- and trisialobiantennary glycans were the major structures. Fucosylation was markedly increased, while O-acetylation of sialic acid was found to be decreased in PH-FN. The alterations in glycosylation and biological activities of FN after PH are different from those of VN, suggesting that these glycoproteins play different biological functions in tissue remodeling.     

Immunological evidence for phenobarbital-stimulated synthesis of cytochrome P-450 in primary cultures of chick embryo hepatocytes

The induction of cytochrome P-450 by phenobarbital was studied in primary cultures of chick embryo hepatocytes. The rate of the de novo synthesis of the induced form of cytochrome P-450 was measured directly and specificially, using form-specific anti-cytochrome antibodies that quantitatively immunoprecipitated this form from the radiolabeled hepatocytes. Additionally, the steady-state levels of the cytochrome were estimated spectrophotometrically and electrophoretically. In the presence of phenobarbital the synthesis of cytochrome P-450_PB by cultured hepatocytes was markedly accelerated. Furthermore, the same cytochrome P-450_PB form was induced by phenobarbital in vivo in chicken liver and in the cultured chick embryo hepatocytes. Their identity was judged from immunological and electrophoretic properties of these induced cytochromes. Immunological cross-reactivity was also detected between the cytochrome P-450_PB forms from chick embryo hepatocytes and from adult rat liver. The immunological cross-reactivity observed between the phenobarbital-induced cytochrome P-450 forms from different species was not observed between the different cytochrome forms with the same liver (Thomas, P.E., Reik, L.M., Ryan, D.E. and Levin, W. (1981) J. Biol. Chem. 256, 1044–1052). Implications as to the evolutionary origin of the different cytochrome forms are discussed.     

Synthesis of fibronectin in normal and osteoarthritic articular cartilage

The content and the biosynthesis of fibronectin was examined in disease-free articular cartilage and in articular cartilage from osteoarthritic canine joints. Fibronectin content was increased in extracts of cartilage from osteoarthritic joints. Incubation of cartilage in vitro with [³H]phenylalanine and subsequent isolation of [³H]fibronectin from a gelatin affinity column and characterization by SDS-polyacrylamide gel electrophoresis and by immunoprecipitation indicated that disease-free and osteoarthritic cartilage explants synthesized fibronectin. About 50% of the [³H]fibronectin was recovered in the incubation medium. The osteoarthritic cartilage synthesized and accumulated up to 5-fold more [³H]fibronectin than disease-free cartilage.     

Fibronectin in the area opaca of the young chick embryo

Summary Distribution of fibronectin-like immunoreactivity was studied in the area opaca of the young chick embryo (stages 4–6 HH) by use of the immunofluorescence and protein A-coupled to colloidal gold techniques. Fibronectin, associated to the basement membrane, formed a fibrillar network, the pattern of which changed from the centre to the periphery of the area opaca. At the ultrastructural level, differences in fibronectin distribution were found between non-moving and moving cells. The epithelial-like cells presented fibronectin staining exclusively on their basal side. Actively migrating cells (edge and mesodermal cells) showed immunoreactive material localized around their entire surface and within the cytoplasm. The fibronectin distribution is discussed in relation to three important phenomena taking place during the early growth of the area opaca: (i) anchorage and migration of the edge cells, (ii) modification of cell shape in relation to mechanical tension, and (iii) expansion of the area vasculosa.     

A large cathepsin D-derived fragment from the central part of the fibronectin subunit chains

A mild cathepsin D digest of fibronectin only contained single-chain peptides of 200, 140 and 70 kDa and double-chain fragments of about 300 and 140 kDa containing the C-terminal disulfide link. Among the single-chain fragments the 200 kDa peptide was a precursor of the 140 kDa and 70 kDa peptides. The latter was correlated to the N-terminal and the former to the central region of the fibronectin subunit chains.     

Extracts of bone contain a potent regulator of bone formation

We prepared aqueous extracts of whole femorae and tibiae of embryonic chicks. An amount of extract containing 25 μg of protein resulted in a 500% increase in DNA synthesis in calvarial cell cultures, and significant effects were detected with 5 μg (55%). The time course for stimulation of DNA synthesis showed a peak occurring 16–20 h after addition of the extract. This matrix factor is nondialyzable, and fractionation on a column of Sephadex G-100 indicated a molecular weight of 60–80 000. At the maximum dose used, [³H]proline incorporation into total protein of calvarial cells was increased by 55%, and thus far, all fractions active in promoting DNA synthesis have been found to increase collagen synthesis in culture chick tibiae. These data are consistent with an effect on osteoblasts as well as bone precursor cells. Extracts prepared from tibiae of 2-day-old chicks, from which the marrow had been removed, also stimulated DNA synthesis (280% increase), thus ruling out the possibility that the factor is a relatively nonspecific mitogen from the hematopoietic cell line. We conclude that bone matrix contains a substance which could regulate bone formation in vitro by control of mitosis in osteogenic precursors and/or stimulation of osteoblast activity.     

Transfer of glucose in the biosynthesis of thyroid glycoproperties I. Inhibition of glucose transfer to oligosaccharide lipids by GDP-mannose

Thyroid rough microsomes catalyzed the synthesis of glucose-containing oligosaccharide lipids which were compared to those extracted from labeled thyroid cells and were found to be largely similar.Glucose transfer to these oligosaccharide lipids in the microsomal system was shown to be markedly depressed by an addition of GDPmannose. This sugar nucleotide, already at 1μM, blocked dolichol-P-glucose synthesis, thus restraining further glucosylation of oligosaccharide lipids. Using this concentration of radioactive GDPmannose in the incubation medium lead to the detection of three glucose containing mannose-labeled oligosaccharide lipids. Double labeling experiments suggested a precursor-product relationship between them.Previously labeled oligosaccharide lipids, containing glucose or not were compared in their efficiency to acr as donors of their oligosaccharide chain to an exogenous synthetic Asn-X-Thr containing peptide. It was foun that the presence of glucose did not signifantly influence the transfer. Free glucose was released during the reaction when using the glucose-labeled oligosaccharide lipid.     

Rapid assembly of gp120 oligosaccharide moieties via one-pot glycosidation-deprotection sequences

Mannosyl trihaloacetimidate donors equipped with a 2-O-Fmoc group can be effectively activated by catalytic Bi(OTf)₃ in glycosidations. Despite the expected participating effect of the Fmoc group, the reaction solvent was found to be decisive for obtaining highly selective α-mannosylations. The Fmoc 2-O-protecting group can be then simply removed from the obtained di-oligosaccharide in the same vessel where the glycosidation is conducted. The resulting oligosaccharide can thus be directly employed as a glycosyl acceptor for further elongation. The preparation of biologically important linear and branched oligomannoses incorporated into HIV gp120 demonstrates that iteration of this one-pot sequence leads to very straightforward oligosaccharide assembly. As an additional result, a rapid approach has been disclosed for accessing a 3,6-OH mannose building-block to be incorporated in branched structures. This relies on a double reductive opening of a di-O-benzylidene mannose intermediate whose regioselectivity appears to be independent of the configuration of the five-membered benzylidene.     

Phaseolus vulgaris phytohemagglutinin contains high-mannose and modified oligosaccharide chains

Phytohemagglutinin, the major lectin in the seeds of the common bean Phaseolus vulgaris L., was isolated by affinity chromatography from cotyledons of nearly mature seeds and from developing cotyledons labeled with [³H]glucosamine, [³H]mannose or [³H]fucose. The protein was subjected to exhaustive proteolysis and the carbohydrate composition of the resulting glycopeptides examined. Two classes of oligosaccharide side-chains were found. The sidechains of the first class are of the high-mannose type, containing two residues of N-acetylglucosamine and 8 or 9 mannose residues. The sidechains of the second class are of the modified type containing N-acetylglucosamine, mannose, fucose, xylose in molar ratios of 2:3.8:0.6:0.5. Two-dimensional gel electrophoresis shows that phytohemagglutinin can be fractionated into seven different glycosylated polypeptides, and that each one contains at least one modified oligosaccharide chain. The results indicate that most glycosylated polypeptides probably contain one chain of each class. The carbohydrate composition of the two types of chains is similar to that found in other plant glycoproteins, but this is the first report of a plant glycoprotein with both highmannose and modified oligosaccharides on the same polypeptide chain.Abbreviations endo H endo--N-acetylglucosaminidase H - GlcN glucosamine - GlcNAc N-acetylglucosamine - Man mannose - PHA phytohemagglutinin This work was done while A.V. was on leave from the Istituto Biosintesi Vegetali, C.N.R., via Bassini 15, I-20133 Milano, Italy     

Monensin inhibits the first cellular movements in early chick embryo

Summary In early chick blastoderm at stage XIII, the interaction of the hypoblast with the epiblast triggers on the epiblast the first extensive cellular migrations, which result in formation of the primitive streak, the source of the axial mesoderm. During this period, extracellular material (ECM) is secreted and assembled into an organized network in the extracellular spaces and is implicated in regulating the behaviour of the cells that contact it. The first cellular migrations and inductions are inhibited when early chick blastoderm is treated with the glycosylation-perturbing ionophore monensin. The difference in amount and in organization of ECM between monensin-treated embryos and control embryos is striking. Even blastoderms at stage X, which are essentially free of ECM, show extensive ECM after monensin treatment. Monensin produces a substantial change in the polypeptide pattern with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from those present in the control embryos. The interference of monensin with the migration and induction mechanisms is permanent in embryos before the primitive streak (PS) stage, and it seems that the respective signals or the sensitivity of the epiblast/hypoblast cells to them must be very stage specific. Monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the hypoblast to interact with the epiblast cells.     

The effects of post-translational processing on dystroglycan synthesis and trafficking

Dystroglycan is a component of the dystrophin glycoprotein complex that is cleaved into two polypeptides by an unidentified protease. To determine the role of post-translational processing on dystroglycan synthesis and trafficking we expressed the dystroglycan precursor and mutants thereof in a heterologous system. A point mutant in the processing site, S655A, prevented proteolytic cleavage but had no effect upon the surface localisation of dystroglycan. Mutation of two N-linked glycosylation sites that flank the cleavage site inhibited proteolytic processing of the precursor. Furthermore, chemical inhibition of N- and O-linked glycosylation interfered with the processing of the precursor and reduced the levels of dystroglycan at the cell surface. Dystroglycan processing was also inhibited by the proteasome inhibitor lactacystin. N-linked glycosylation is a prerequisite for efficient proteolytic processing and cleavage and glycosylation are dispensable for cell surface targeting of dystroglycan.     

Role of the cellular attachment domain of fibronectin in the phagocytosis of beads by human gingival fibroblasts in vitro

Summary To study the role of phagocytosis in periodontal tissues, internalization of fibronectin-coated latex beads by Gin-1 fibroblast populations was investigated. Demonstration of phagocytosis by internalization of beads was confirmed by immunofluorescence microscopy, electron microscopy, and flow-cytometry. The percent of cells phagocytosing beads measured by flow-cytometry was negligible at 4° and 23°C, but increased to approximately 17% at 37°C. As measured by automated image analysis, the percentage of phagocytosing cells increased linearly from 8 to 22 with increasing fibronectin concentration of the incubation solution from 30 ng to 300 g/ml. Similar linear increases in the percentage of phagocytosing cells were observed when beads were incubated with cells for periods ranging from 2 h to 2 days. To examine the role of the Arg-Gly-Asp receptor in mediating phagocytosis, fibronectin-coated beads were first coated with either Gly-Arg-Gly-Asp-Ser-Pro or Gly-Arg-Gly-Glu-Ser-Pro peptides at concentrations of 0.125, 0.5, and 1 mg/ml, or with control vehicle, and then incubated with cells. Phagocytosis was completely blocked at 1 mg/ml of the Gly-Arg-Gly-Asp-Ser-Pro peptide, but the Gly-Arg-Gly-Glu-Ser-Pro peptide showed no significant inhibition compared to control values. Blocking antibodies to the cell attachment domain of the fibronectin molecule also reduced the percentage of phagocytosing cells significantly. The data show that these phagocytic assays are sensitive enough to detect the influence of incubation temperature and time, cellular heterogeneity, ligand type, and ligand concentration on the percentage of phagocytosing cells. Further, the mechanisms which determine internalization of fibronectin-coated beads rely in part on the initial binding of ligand to the Arg-Gly-Asp receptor present on fibroblasts.     

A convergent strategy for the preparation of N-glycan core di-, tri-, and pentasaccharide thioaldoses for the site-specific glycosylation of peptides and proteins bearing free cysteines

Mammalian glycoprotein biosynthesis produces heterogeneous ranges of proteins that possess the same peptide backbone but differ in the nature and site of glycosylation. This feature has frustrated efforts to develop therapeutic glycoproteins as well as the elucidation of biological functions of individual glycoforms. We have developed an attractive approach to well-defined glycoforms of glycoproteins by oxidative coupling of thioaldoses to cysteine-containing peptides and proteins to give disulfide-linked neoglycoconjugates. To this end, the chemical synthesis di-, tri-, and pentasaccharide N-glycan thioaldoses was undertaken. A convergent approach was used for the preparation of the pentasaccharide containing a 'synthetically difficult' beta-mannoside linkage. This linkage was installed by forming initially the corresponding beta-glucoside-containing pentasaccharide, followed by inversion of configuration at C-2. This approach exploited a levulonyl ester at C-2 of a glucosyl donor, which directed the coupling to give the beta-glucoside exclusively and could be removed selectively using hydrazine acetate without affecting other base-labile functionalities. The resulting alcohol was converted into a triflate, which was displaced by tri-n-butylammonium acetate to give a beta-mannosidic linkage. The trisaccharide N-glycan was prepared in a similar manner. Thioaldoses were prepared by displacing the peracetylated alpha-glycosyl chlorides with thioacetate to give the peracetylated beta-thioacetates, which upon saponification gave the desired compounds. The incubation of molar excesses of chitobiose thioaldose with cysteine-containing glutathione and BSA resulted in the site-specific formation of a disulfide-linked neoglycopeptide and neoglycoprotein, respectively.     

Changes in protein glycosylation during chick embryo development

To investigate the molecular changes in cell-surface glycoproteins during chick embryo development, fibroblasts from 8- and 16-day embryos were extensively digested by pronase after (i) metabolic labeling with radioactive precursors and (ii) external labeling. Two main classes of glycopeptide pronase digestion product were distinguished by Sephadex G-50 column chromatography. The large material excluded was mostly composed of glycosaminoglycans. The small retarded glycopeptides underwent age-related modifications. Those in the 8-day cells were mainly N-linked, whereas 16-day cells contained both O- and N-linked glycopeptides. The evolution of high-mannose chains in younger cells to complex-type chains in the older cells is suggested by (i) the decrease in the mannose-to-galactose and mannose-to-N-acetylglucosamine ratio with embryo development, and (ii) the fact that endo-β-N-acetylglucosaminidase H treatment released more oligomannosyls from younger than from older embryo cell glycopeptides. Small glycopeptides were also more highly sialylated in 16-day cells than in 8-day cells. The present results provide the first biochemical evidence that both quantitative and qualitative modifications occur in cell-surface glycoconjugates during the late stages of chick embryo development.     

A structural study of the asparagine-linked oligosaccharide moiety of duck ovomucoid

Asparagine-linked oligosaccharides of duck ovomucoid were released quantitatively from the protein by digestion with glycoamidase A (from almond), the reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated using two different types of high performance liquid chromatography (HPLC) on a reversed phase column and an amide adsorption column. More than sixteen different oligosaccharides were separated and the structures were characterized by a combination of the 2-dimensional sugar mapping technique using HPLC, exoglycosidase digestion, and proton nuclear magnetic resonance measurements (1- and 2-dimensional). Furthermore, the HPLC profile of duck ovomucoid oligosaccharides was compared with previously reported profiles obtained from quail and chicken ovomucoids.Abbreviations COSY chemical shift-correlated spectroscopy - DQF-COSY double quantum filtered COSY - DSS sodium, 4,4-dimethyl-4-silapentane 1-sulfonate - Gal D-galactose - GlcNAc or GN N-acetyl-D-glucosamine - HOHAHA homonuclear Hartmann-Hahn spectroscopy - Man or M D-mannose - NOE nuclear Overhauser enhancement - ODS octadecylsilyl - PA pyridylamino - ROESY rotating frame nuclear Overhauser effect spectroscopy - SDS/PAGE sodium dodecyl sulfate/polyacrylamide gel electrophoresis