Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specific subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both thein vitro andin vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles,in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greaterin vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with both2,3 and2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greaterin vitro biological potency compared to those of the pituitary human follicle stimulating hormone.     

Glycosylation pattern and processing of envelope gene products encoded by glycosylation mutants of Friend spleen focus-forming virus

The protein encoded by the envelope gene of Friend spleen focus-formingvirus is responsible for the acute leukaemogenicity of thisvirus. In order to correlate glycosylation and intracellularprocessing of this protein with viral pathogenicity, envelopegene products of pathogenic and apathogenic glycosylation mutantswere expressed in Rat-1 cells and metabolically labelled with[6-³H] glucosamine. Following immunoprecipitation, primary andsecondary gene products (gp55, gp65) were separated by preparativepolyacrylamide gel electrophoresis. Oligosaccharides were releasedfrom tryptic glycopeptides by treatment with endo-ß-N-acetylglucosaminidaseH (gp55), peptide-N⁴-(N-acetyl-ß-glucosaminyl)asparagineamidase F (gp65) or by reductive ß-elimination. Resultingglycans were characterized by cochromatography with authenticoligosaccharide standards using different HPLC systems and digestionwith exoglycosidases. The results revealed that the primaryenvelope gene products of pathogenic glycosylation mutants were,in part, further processed in Rat-1 cells similar to wild-typeglycoprotein, resulting in polypeptides carrying complex-typeN-glycans as well as partially sialylated O-linked oligosaccharides.In contrast, corresponding glycoproteins encoded by apathogenicmutants were found to remain at the level of the primary translationproduct exclusively comprising high-mannose-type N-glycans.Hence, intracellular maturation of the envelope gene productsin this model cell line seems to correlate with the in vivopathogenicityof the glycosylation mutants studied. carbohydrate structure glycoprotein murine leukaemia virus oligosaccharide processing SFFV     

Establishment of in vitro Cultures of Tapinanthus bangwensis (Engler and K. Krause) Danser and the Effects of Sugars on Growth

Culturing of organs in vitro has been successfully employedin studies on morphogenesis and nutritional requirements ofparasitic and semi-paraaitic angiosperms. Tapinanthus bangwensis,a semi-parasite, has been successfully cultured on chemicallydefined media. By and large the parasite will thrive well ina medium of mineral salts and sucrose at its optimal concentration(4 per cent). However, the parasite is able to metabolize awide range of sugars most of which show similar concentrationoptima Although the growth in vivo was simulated in vitro inthe early stages, it was found that in the later stages growthin vitro was much slower than growth in vivo. The growth differencesobserved in the different media may reflect some of the physiologicaldifferences that are responsible for the selective nature ofthe parasite's development and establishment on different hosts     

Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli

Background  

The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity.     

Characterization of ganglioside associated with the thyrotrophin receptor

The receptor protein for thyrotrophin (thyroid-stimulating hormone;TSH) is associated with a glycosphingolipid moiety. The proteinbelongs to the family of receptors that couple to guanine nucleotidebinding proteins; the glycosphingolipid contains sialic acidand belongs to the family of gangliosides. This report definesthe structure of the receptor ganglioside in the Fisher ratthyroid cell line (FRTL-5). Receptor protein was purified byTSH affinity chromatography from FRTL-5 cells, biosyntheticallylabelled with [³H]galactose and [³H]glucosamine, and resolvedby SDS-PAGE. A single radiolabelled band of M_r     

Novel Asn-linked oligosaccharides terminating in GaINAc{beta}(1->4)[Fuca(1->3)]GlcNAcB(1->{bullet}) are present in recombinant human Protein C expressed in human kidney 293 cells

Recombinant human Protein C (rHPC), expressed in human kidney293 cells, has a higher anticoagulant activity than plasma HPC,while its in vivo circulatory half-life is essentially unalteredcompared to that of the natural protein. In seeking to elucidatethe molecular basis for the improved efficacy of the recombinantantithrombotic drug, we focused on the carbohydrate moiety ofrHPC. Protein C is a heavily post-translationally modified serineprotease with four N-glycosylation sites. Glycosyl compositionanalysis of rHPC revealed a 5-fold higher fucose content anda 2-fold lower sialic acid content compared to plasma HPC. Inaddition, we found that rHPC contains N-acetylgaiac-tosamine(2.6 mol GalNAc/mol rHPC) in its Asn-linked oligosaccharides,while plasma HPC is devoid of GalNAc. The Asn-linked oligosaccharidesof rHPC were released by N-glycanase and separated into 25 fractionsby high-pH anion-exchange chromatography. The most abundantoligosaccharides were structurally characterized by glycosylcomposition and linkage analysis, in conjunction with ¹H-NMRspectroscopy at 600 MHz. The structure of the major neutraloligosaccharide in rHPC was determined to be: Two representatives of the sialylated oligosaccharides in rHPCare: and Thus, many of the Asn-linked oligosaccharides in rHPC were foundto terminate in GaINAcß(1     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

Suppression of allogeneic reactivity in vitm by the syncytiotrophoblast membrane glycocalyx of the human term placenta is carbohydrate dependent

Immunosuppressive factors isolated from trophoblast are knownto block both innate and major histocompatability complex (MHC)-dependentcell-mediated immune responses in vitro and, in some cases,in vivo. We investigated the biochemical nature of these factors,which is presently unknown. Immunosuppressive activity, assessedby inhibition of two-way MLR, was extracted from term syncytiotrophoblastmicrovilli using 3 M KCl. The activity resisted both extensivepronase digestion and heating to 90°C for 1 h, demonstratingthat intact membrane proteins were not required. Although purifiedprotein-linked oligosaccharides released by hydrazinolysis fromthe syncytiotrophoblast membrane were themselves inactive, theyblocked the immunosuppressive activity of the KCl extract. Afterpronase digestion, the activity could be fractionated by TSK55S gel filtration, followed by C18 reverse-phase chromatography.Sequential exoglycosidase digestion of hydrazine-released sugarsof the active fraction demonstrated that it contained neutralN-linked oligomannose and hybrid oligosaccharides, which normallymake up <3% of the total syncytiotrophoblast-derived proteinglycan Library. These glycopeptides of the active fraction wereassociated with membrane phospholipid micelles. The possiblemechanism by which incompletely processed N-linked oligosaccharidesexpressed by a variety of syncytiotrophoblast membrane glycoproteinsmay block allogeneic reactivity when presented as polyvalentsugar groups is discussed. glycoprotein immunosuppression oligosaccharides pregnancy trophoblast     

Structural analysis of hexa to dodecaoligosaccharide-alditols released by reductive {beta}-elimination from oviducal mucins of Bufo bufo

Hexa to dodecasaccharide-alditols of the jelly coat surroundingthe eggs of the toad Bufo bufo were studied by methylation analysis,MALDI-TOF mass spectrometry, and ¹H-NMR spectroscopy. Thesehighly species-specific carbohydrate chains exhibit new structuralfeatures, such as the elongation of the blood group A determinantwith an external -1,3-linked galactose unit, or ramificationbelonging from a fucosylated galactose. The most representativeoligosaccharide-alditol of the series was defined as following: Since the jellies surrounding amphibian eggs are involved inegg-sperm interactions, these structural investigations canprovide biochemical support for exploring the fertilizationprocess. amphibian egg jelly coats Bufo bufo NMR oligosaccharides     

Characterization of asparagine-linked oligosaccharides on a mouse submandibular mucin

The asparagine-linked oligosaccharides from an adult femalemouse submandibular gland mucin were released by treatment withpeptide-N⁴-(N-acetyl-ß-glucosaminyl)asparagine amidaseF or endo-ß-N-acetylglucosaminidase H. Endo-ß-N-acetylglucosaminidaseH appeared to be more effective at releasing the asparagine-linkedoligosaccharides from this mucin than was peptide-N⁴-(N-acetyl-ß-glucosaminyl)-asparagineamidase F. After quantitative reductive labelling with the fluorophore,8-aminonaphthalene-1, 3, 6-sulphonic acid, the oligosaccharideswere separated by polyacrylamide gel electrophoresis and isolated.The individual oligosaccharides were sequenced by a batteryof recombinant exoglycosidases. Approximately 50% of the oligosaccharideswere of the high-mannose type. The five-mannose member of thisfamily was the most prevalent. The second group of oligosaccharideswere of the non-bisected hybrid type. No complex asparagine-linkedoligosaccharides were detected. The hybrids exhibited both biantennaryand triantennary branching patterns. The triantennary hybridwas the most common hybrid at >30% of all oligosaccharides.With 98% of the hybrid oligosaccharides sialylated and all lackinga bisecting N-acetylglucosamine, these oligosaccharides as agroup have been only rarely observed in other glycoproteins.The fully sialylated triantennary hybrid may be unique. asparagine-linked oligosaccharides biantennary salivary mucin sialylated hybrid triantennary     

The Effects of Griseofulvin on Certain Phytopathogenic Fungi

In vitro and in vivo investigations have been carried out intothe reaction of actively growing hyphae of Botrytis spp. onencountering an environment containing griseofulvin. In vitro, the reactions noted were slowing of the growth rate,stimulation to branching, abnormal appearance of hyphal protoplasm,and loss of rigidity of the hyphal membrane in the region ofthe tip. The antibiotic is not translocated within the mycelium. In vivo, Botrytis tulipae was incapable of penetrating cellmembranes in the stem of tulips watered with griseofulvin. Theantibiotic was shown to delay or to prohibit the penetrationof onion epithelium by B. allii, depending on concentration.The characteristic curling and stunting of hyphae caused bygriseofulvin have both been observed to occur in plant cells.     

Anti-Influenza Neuraminidase Inhibitor Oseltamivir Phosphate Induces Canine Mammary Cancer Cell Aggressiveness

Oseltamivir phosphate is a widely used anti-influenza sialidase inhibitor. Sialylation, governed by sialyltransferases and sialidases, is strongly implicated in the oncogenesis and progression of breast cancer. In this study we evaluated the biological behavior of canine mammary tumor cells upon oseltamivir phosphate treatment (a sialidase inhibitor) in vitro and in vivo. Our in vitro results showed that oseltamivir phosphate impairs sialidase activity leading to increased sialylation in CMA07 and CMT-U27 canine mammary cancer cells. Surprisingly, oseltamivir phosphate stimulated, CMT-U27 cell migration and invasion capacity in vitro, in a dose-dependent manner. CMT-U27 tumors xenograft of oseltamivir phosphate-treated nude mice showed increased sialylation, namely α2,6 terminal structures and SLe(x) expression. Remarkably, a trend towards increased lung metastases was observed in oseltamivir phosphate-treated nude mice. Taken together, our findings revealed that oseltamivir impairs canine mammary cancer cell sialidase activity, altering the sialylation pattern of canine mammary tumors, and leading, surprisingly, to in vitro and in vivo increased mammary tumor aggressiveness.     

Structural characterization of novel oligosaccharides of cell-surface glycoproteins of Trypanosoma cruzi

Affinity-purified glycopeptides were prepared from Trypanosomacruzi using the carbohydrate-specific monoclonal antibody WIC29.26.These glycopeptides contain rhamnose, fucose, xylose, and galactose,in the ratio 1:1:2:3. A series of oligosaccharides was releasedfrom the glycopeptides by mild acid hydrolysis, while, in contrast,no oligosaccharides were released by either peptide N-glycosidaseF or conventional base-catalyzed ß-elimination andreduction. This suggested the presence of a phosphodiester linkagebetween the carbohydrate and peptide, which was further supportedby the detection of phosphothreonine in the glycopeptides. Themild acid liberated (MAL) fraction was resolved into two majoracidic oligosaccharides (MAL-P1 and MAL-P2), two minor neutraloligosaccharides (MAL P1b and MAL-P2b) and a neutral fraction(MAL-N1), consisting of Gal and Xyl monosaccharides. The MAL-P1and MAL-P2 oligosaccharides proved to be hexa- and heptasaccharidesthat shared a common xylose reducing terminus, but differedby one galactofuranose residue, and their negative charge wasshown to be due to the presence of cyclic-phosphate attachedto nonreducing terminal galactofuranose residues. The MAL-P1band MAL-P2b oligosaccharides appeared to be nonphosphorylatedversions of MAL-P1 and MAL-P2. Partial structures of MAL-P1and MAL-P2 are suggested, based on compositional analyses, electrospraymass spectrometry, and tandem mass spectrometry before and afterpermethylation. The origin and significance of these uniquetrypanosomatid glycoconjugates is discussed. glycoprotein monoclonal antibody oligosaccharide structure Trapanosoma cruzi     

Differential expression of an endogenous mannose-binding protein R1 during muscle development and regeneration delineating its role in myoblast fusion

The role of the endogenous brain carbohydrate-binding proteinR1 in muscle cell development and regeneration was analysedboth in vivo and in vitro. In vivo, R1 was developmentally regulated,with an embryonic 65 000 subunit and a neonatal 67 000 subunit,being replaced progressively by a 135 000 adult form. LectinR1 was intracellularly localized at birth and in the prenatalperiod. During development and at the time of myoblast fusion,the antigen was progressively found at the surface, where itremained at low levels in the adult. In vitro, in pure myoblastcultures, only the embryonic form was present. The ultrastructuralstudies indicated that the lectin could participate in the membranefusion process during myoblast fusion. The specific role inmyoblast fusion, derived from the ultrastructural localizationof R1, was evidenced by a strong inhibitory effect of anti-R1 Fab fragments (10–100 µg/ml), relative to controlFab fragments. In vivo, the embryonic subunit pattern and subcellulardistribution of R1 reappeared in muscle cells after lesion ofthe adult muscle. This suggested that, as observed in vitro,R1 participated in vivo in the phenomenon of myoblast fusion.Similar modifications in subunit expression were observed inmuscles after denemation (the embryonic form of lectin R1 reappearingafter lesion), suggesting that R1 could be involved in the processof neuromuscular junction formation. Thus, it is proposed thatthe carbohydrate-binding protein R1 is an important recognitionmolecule for the formation of myotubes. Its potential involvementin a recognition process between axons and muscle cells duringneuromuscular junction formation is discussed. culture development fusion N-glycan glycoprotein lectin mannose myoblast     

A 23 kDa membrane glycoprotein bearing NeuNAc{alpha}2-3Gal{beta}1-3GalNAc O-linked carbohydrate chains acts as a receptor for Streptococcus sanguis OMZ 9 on human buccal epithelial cells

Streptococcus sanguis colonizes several human oral surfaces,including both hard and soft tissues. Large salivary mucin likeglycoproteins bearing sialic acid residues are known to bindvarious S.sanguis strains. However, the molecular basis forthe adhesion of S.sanguis to human buccal epithelial cells (HBEC)has not been established. The present study shows that S.sanguisOMZ 9 binds to exfoliated HBEC in a sialic acid-sensitive manner.The desialylation of such cells invariably abolhhes adhesionof S.sanguis OMZ 9 to the cell surface. A soluble glycopeptidebearing short sialylated O-linked carbohydrate chains behavesas a potent inhibitor of the attachment of S.sanguis OMZ 9 toexfoliated HBEC. The resialylation of desialylated HBEC withCMP-sialic acid and Galß1,3GalNAc     

Structures of the carbohydrate moiety attached to one site in the first domain of turkey ovomucoid: elucidation by 1H-n.m.r. spectroscopy

1H-N.m.r. spectroscopy was used to elucidate the primary structures of the carbohydrate moiety attached to asparagine at residue 53 in the first domain of turkey ovomucoid, a serine proteinase inhibitor. The carbohydrate moiety is a heterogeneous mixture of three structurally closely related complex-type oligosaccharides. Of the total carbohydrate moiety, 61% is tetra-antennary with terminal galactose and with an intersecting N-acetylglucosamine, and containing an additional N-acetylglucosamine (10') attached to mannose (4'). Another 23% is tri-antennary with terminal galactose and with an intersecting N-acetylglucosamine. The remaining 16% is tri-antennary with terminal galactose (6 and 8 only), with an intersecting N-acetylglucosamine.     

Glycosylation of catalase inhibitor necessary for activity

A protein isolated from maize scutella which inhibits catalase in vitro has been shown to contain 12% carbohydrate in the form of galactose. This corresponds to four galactose molecules per inhibitor subunit. Removal of the carbohydrate with β-galactosidase or blockage with a galactose-specific lectin abolished activity of the inhibitor.     

Aluminium Effects on ATPase Activity and Lipid Composition of Plasma Membranes in Sugar Beet Roots

The effect of aluminium (Al) in vivo and in vitro on root plasmamembranes has been studied in two sugar beet (Beta vulgarisL.) cultivars, Monohill (Al-sensitive) and Regina (relativelyAl-tolerant). Although Al in vitro inhibited the MgATPase inan uncompetitive way for both cultivars raised in the absenceof Al, the specific K⁺-activation of the MgATPase was only inhibitedby Al in cv. Monohill. Arrhenius analysis of the MgATPase activity showed that theeffect of Al in vitro depended on whether or not the plantswere exposed to Al in vivo. Al treatment in vitro of the MgATPasefrom control plants cultivated at a low pH (5·4) causedan increase in the phase transition temperature from 17 to 22°C. Only at a higher pH range (pH 6·1) could a secondtransition temperature be induced (at 9 °C). By additionof Al in vitro to plants cultivated with Al at pH 5·4,the slopes of the activity plots did not change. Aluminium changedthe K_m of the ATPase for MgATP in an opposite way by treatmentin vivo and in vitro. Lipid analyses of the plasma membranes showed that the acylcomposition differed little following Al treatment in vivo,but that the ratio of phosphatidylcholine: phosphatidylethanolamineincreased. The changes correlated with the observed change inthe K_m for the MgATPase. We conclude that the main effect ofAl on the MgATPase is not due to the formation of an Al-ATPcomplex. Instead, Al may bind to the membrane-bound enzyme(s)and/or modify the lipid environment. Key words: Aluminium, ATPase, Beta vulgaris, lipids     

Defoliation and its Effects on Pod and Seed Development in Oil Seed Rape (Brassica napus L.)

Both in vivo and in vitro techniques have been used to followthe development of individual pods on the terminal inflorescenceof undefoliated and defoliated plants of oil-seed rape (Brassicanapus cv. Maris Haplona). For any pod, a rapid increase in podlength occurred between 2 d and 8 d after flower opening andthis preceded by approximately 2 d the increase in pod width,the rate of which was less than that for length. An increasein the diameter of individual seeds coincided with the increasein pod width. Regional increases in the length and width ofpods were associated with the presence of developing seeds inthese regions. Key words: Brassica napus L., Development, In vivo, Pod and seed, Stress