Alterations of O-glycan biosynthesis in human colon cancer tissues

Human colon cancer is associated with antigenic and structuralchanges in mucin-type carbohydrate chains (O-glycans). To elucidatethe control of the biosynthesis of these O-glycans in coloncancer, we have studied glycosyltransferase and sulphotransferaseactivities involved in the assembly of elongated O-glycan structures.We analysed homogenates prepared from cancer tissue, adjacentnormal and distal normal tissue from 20 patients. Several transferaseactivities showed pronounced changes in cancer tissue. The changescorrelate with previous findings of a loss of O-glycans in cancermucins, but did not always correlate with levels of Tn, sialyl-Tn,T and Le^x antigens in homogenates or with the differentiationstatus and Duke's stages of the cancer tissue or the patient'sblood type, sex and age. UDP-GlcNAc: Gal NAc-R ß3-N-acetylglucosaminyltransferase(where GlcNAc is N-acetyl-D-glucosamine and GalNAc is N-acetyl-D-galactosamine)synthesizing O-glycan core 3, GlcNAcß1-3GalNAc-, CMP-sialicacid: GalNAc-peptide     

重组人可溶性PDGFRβ/Fc在昆虫细胞Sf9中的表达

【目的】利用昆虫细胞Bac-to-Bac杆状病毒表达系统表达血小板源性生长因子受体β (PDGFRβ)链膜外区与人IgG Fc片段的可溶性受体融合蛋白sPDGFRβ/Fc,并检测重组蛋白的特异性和生物活性。【方法】采用Bac-to-Bac系统,构建重组转移质粒pFastbac-sPDGFRβ/Fc,转化到含穿梭载体Bacmid的感受态细胞DH10Bac中,使目的基因与杆状病毒基因组DNA发生位点特异性重组,获得重组病毒DNA,将其通过脂质体转染昆虫细胞Sf9获得重组病毒。将该重组病毒感染Sf9无血清细胞系,在Sf9细胞中表达sPDGFRβ/Fc,对表达产物进行Western blotting检测和Protein A亲合层析纯化,并进一步通过MTT法检测获得的重组蛋白生物学活性。【结果】重组病毒感染Sf9细胞后,经Western blotting分析,能检测到一条分子量约为97 kDa的特异性条带,与目的蛋白大小相符。通过Protein A亲和层析,获得了纯度达75％以上,表达量为1 μg/mL细胞培养上清的重组融合蛋白,MTT结果显示该重组融合蛋白sPDGFRβ/Fc具有抑制PDGF刺激的Balb/c 3T3细胞增殖的能力。【结论】具有生物活性的重组可溶性受体融合蛋白sPDGFRβ/Fc可在昆虫细胞中成功地得到表达。     

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     

野桑蚕GST-Omega1基因在草地贪夜蛾Sf9细胞中的表达

谷胱甘肽 S-转移酶（glutathione S-transferases,GSTs）是昆虫的重要解毒酶之一。为了研究野桑蚕Bombyx mandarina中谷胱甘肽S-转移酶在真核表达系统中的表达情况。本研究通过RT-PCR从野桑蚕中肠中获得GST-Omega1基因的cDNA序列,该基因的开放读码框为771 bp,编码256 个氨基酸。对推导的氨基酸序列用NCBI的蛋白质Conserved Domains工具进行在线分析,结果显示GST-Omega1的氨基酸序列中具有Cys38和8个GSH结合位点的Omega类基因保守序列。对所获得的基因克隆进表达载体pFastBacHT b中获得pFast-GST-Omega1,将其转化DH10Bac感受态细胞,获得Bac-GST-Omega1重组病毒DNA,用脂质体法转染草地贪夜蛾Sf9细胞,获得重组病毒。对表达产物经SDS-PAGE和Western blotting分析,能检测到一条分子量约为33 kD的特异性条带,与推导的融合蛋白大小相符,该目的蛋白的表达量占总蛋白的14.4%。目的蛋白经His·Bind树脂纯化,用Lineweaver Burk作图法测定其K_m和V_max,结果显示其K_m为2.81 µmol/L,V_max为2.70 µmol/(mg·min）。     

Processing of asparagine-linked oligosaccharides in insect cells. N-Acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells

The levels of ß1,2-N-acetylglucosaminyltransferase(GlcNAc-T) I and II activities in cultured cells from Bombyxmori(Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda(Sf-9 and Sf-21) were investigated. Apart from initial experimentswith Man     

The N-glycosylation defect of cwh8Delta yeast cells causes a distinct defect in sphingolipid biosynthesis

CWH8/YGR036c of Saccharomyces cerevisiae has been identifiedas a dolichylpyrophosphate (Dol-PP) phosphatase that removesa phosphate from the Dol-PP generated by the oligosaccharyltransferase(OST), while it adds N-glycans to nascent glycoproteins in theendoplasmic reticulum (ER). Lack of CWH8 was proposed to interruptthe so called dolichol (Dol) cycle by trapping Dol in the formof Dol-PP in the ER lumen. Indeed, cwh8D mutants display a severedeficiency in N-glycosylation. We find that cwh8D mutants havestrongly reduced levels of inositolphosphorylceramide (IPC),whereas its derivative, mannosyl-(inositol-P)₂-ceramide (M(IP)₂C)is not affected. Microsomes of cwh8D contain normal ceramidesynthase and IPC synthesis activities. Within a large panelof mutants affecting Dol dependent pathways such as N- or O-glycosylation,or glycosylphosphatidyl inositol (GPI)-anchoring, only the mutantshaving a deficiency of N-glycan addition show the defect inIPC biosynthesis. By mutating genes required for the additionof N-glycans or by treating cells with tunicamycin (Tm) onecan similarly reduce the steady state level of IPC and exactlyreproduce the phenotype of cwh8D cells. Some potential mechanismsby which the lack of N-glycans could lead to the sphingolipidabnormality were further explored.     

Oligosaccharide profiles of HIV-2 external envelope glycoprotein: dependence on host cells and virus isolates

The glycosylation pattern of the external envelope glycoproteinof human immunodeficiency virus type 2 (HIV-2) was studied independence on host cells and virus isolates. Strains HIV-2_ALT,HIV-2_ROD and HIV-2_D194, differing in their biological propertiesand in the amino acid sequences of their env genes, were propagatedin MOLT4, HUT78 and U937 cells, in human peripheral blood lymphocytesand monocytes/macrophages in the presence of [6-³] glucosamine.Radiolabelled viral glycoproteins were isolated from the cell-freesupernatants and digested with trypsin. Glycans were sequentiallyliberated by endo-ß-N-acetylglucosaminidase H andpeptide-N⁴-(N-acetyl-ß-glucosaminyl) asparagine amidaseF, and fractionated according to charge and size. Comparisonof the oligosaccharide profiles revealed that the envelope glycoproteinsof different virus isolates, propagated in the same host cells,yielded very similar glycan patterns, whereas cultivation ofan isolate in different host cells resulted in markedly divergentoligosaccharide maps. Variations concerned the proportion ofhigh-mannose-, hybrid- and complex-type substituents, as wellas the state of charge and structural parameters of the complex-typespecies. As a characteristic feature, complex-type glycans ofmacrophage-derived viral glycoprotein were almost exclusivelysubstituted by lactosamine repeats. Hence, glycosylation ofthe HIV-2 external envelope glycoprotein seems to be primarilygoverned by host cell-specific factors rather than by the aminoacid sequence of the corresponding polypeptide backbone. envelope glycoprotein glycosylation human immunodeficiency virus type 2     

The yeast CWH41 gene encodes glucosidase I

N-Glycosylation in the yeast Saccharomyces cerevisiae entailsthe synthesis of a Glc₃Man₉GlcNAc₂ oligosaccharide precursorwhich is subsequently transferred to suitable protein acceptors,a process which is conserved among all eukaryotes. Processingof the oligosaccharide occurs immediately following this transfer,the first step being the removal of the terminal     

Human non-secretory ribonucleases. II. Structural characterization of theN-glycans of the kidney, liver and spleen enzymes by NMR spectroscopy and electrospray mass spectrometry

The N-glylycans have been removed by peptide-N-glycosidase F(PNGase F) from purified human non-secretory RNases derivedfrom kidney, liver and spleen. The spleen RNase was purifiedby two procedures, one of which did not include the usual acidtreatment step (0.25 M H₂SO₄, 45 min, 4C), to determine ifacid treatment alters the carbohydrate moieties. TheN-glycansof the RNases were fractionated by Bio-Gel P-4 chromatographyand analysed by 600 MHz ¹H-NMR spectroscopy and electrospraymass spectrometry. All four non-secretory RNase preparationscontained the following structures: The relative amounts of the trisaccharide, pentasaccharide andhexasaccharide appeared to vary slightly in the different tissueRNases. The overall results indicate: (i) that acid treatmentduring purification does not alter the N-glycans of non-secretoryRNases; (ii) that the N-glycans from kidney, liver and spleennon-secretory RNases are very similar, if not identical, toone another, but different from the N-glycan structures reportedfor secretory RNase. N-glycans non-secretory RNases     

意大利蜜蜂蜂毒磷脂酶A2基因在杆状病毒-昆虫细胞系统中的表达

利用Bac to Bac系统将意大利蜜蜂蜂毒磷脂酶A₂（AmPLA₂）基因cDNA克隆至转移载体pFastBacHTa中,得到pBacHT-AmPLA₂,再将其转化入含穿梭载体Bacmid的受体大肠杆菌DH10Bac中,通过转座作用,得到含AmPLA₂基因的重组病毒rBacmid-AmPLA₂的DNA。提取其基因组DNA,用脂质体介导转染粉纹夜蛾细胞Tn-5B1-4,得到重组病毒rACV-Bac-AmPLA₂。用此重组病毒感染Tn-5B1-4细胞, 在细胞中表达AmPLA₂。SDS-PAGE电泳结果显示,与6×His Tag融合表达的产物蛋白分子量约为18 kD左右,表达量约占细胞总蛋白的5.35%。Western blot印迹显示,融合表达产物能与意大利蜜蜂蜂毒AmPLA₂抗血清发生免疫反应。生物活性测定显示,含表达产物的细胞蛋白粗提物对底物蛋黄的酶活力约为6.13 μmol·min^-1·mg^-1。     

Glycosidases from Cotyledons of Pisum sativum L.

-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH₄)₂SO₄, and chromatography on ConcanavalinA¹-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.0–4.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful.     

Immunogenicity and safety of H1N1 influenza hemagglutinin protein expressed in a baculovirus system

Although most influenza vaccines are produced in eggs, new types of vaccines must be developed. In this study, the immunogenicity and safety of a baculovirus‐expressed hemagglutinin (HA) of H1N1 influenza virus (Korea/01/2009; designated “HA‐Bac‐K”) was compared with those of a commercially available baculovirus‐expressed HA (designated “HA‐Bac‐C”) and an Escherichia coli‐expressed HA (designated “HA‐E. Coli‐K”). HA‐Bac‐K succeeded in inducing hemagglutination inhibition and neutralization antibodies in mouse and ferret models. The different immunogenicities observed may be attributable to the different expression systems and purification protocols used. Our work suggests that HA expressed in a baculovirus system is an effective and safe candidate influenza vaccine.     

Expression of rice gall dwarf virus outer coat protein gene (<Emphasis Type="Italic">S8</Emphasis>) in insect cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48–72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.     

Effects of Fluorogibberellins on Plant Growth and Gibberellin 3r{beta}-Hydroxylases

3rß-Fluorogibberellin A₉ (3rß-fluoro-GA₉),3rßfluoro-GA₂₀, 3rß-fluorodeoxygibberellinC (3rß-fluoro-DGC) and 13-fluoro-GA₉ were prepared,and their effects on plant growth and gibberellin (GA) 3rß-hydroxyIaseswere examined. 3rß-Fluoro-GA₉ and 3rß-fluoro-GA₂₀promoted the growth of dwarf rice (Oryza sativa L. cv. Tan-ginbozu)seedlings to three times higher than the control seedlings ata dosage of 3 µ plant^–1, and 3rßfluoro-DGCto twice higher at the same dosage. 3rßg-Fluoro-GA₉was active in cucumber (Cucumis sativus L.) hypocotyl assay,its activity being about one-thirtieth as much as that of GA₄.3rß-Fluoro-GAs were active per se in promoting theshoot elongation of rice. 3rß-Fluoro-DGC inhibitedthe 3rß-hydroxylation of [³H₂]GA₉ to [³H]GA₄ by GArß-hydroxylase from bean (Phaseolus vulgaris L.),but 3rß-fluoro-GA₉ and 3rß-fluoro-GA₂₀ didnot show any effects on the enzyme activity. These 3rß-fluoro-GAsalso showed no or only a weak inhibitory effect on the rß-hydroxylasefrom pumpkin (Cucurbita maxima L.). 13-Fluoro-GA₉ promoted growthof rice and cucumber seedlings, and inhibited the 3rß-hydroxylasesfrom both bean and cucumber. 13-Fluoro-GA₉was converted into13-fluoro-GA₄ and 2,3-didehydro-13-fluoro-GA₉, in a cell-freesystem from bean, and conversion of 13-fluoro-GA₉ into 13-fluoro-GA₄was also observed in a cell-free system from pumpkin. Theseresults suggest that 13-fluoro-GA₉ is one of the substratesof GA 3rß-hydroxy-lases, and that 13-fluoro-GA₉ isactive as a result of the conversion to 13-fluoro-GA₄ in riceand cucumber seedlings. (Received October 27, 1997; Accepted March 13, 1998)     

Isolation, characterization and inactivation of the mouse Mgat3 gene: the bisecting N-acetylglucosamine in asparagine-linked oligosaccharides appears dispensable for viability and reproduction

The biosynthesis of complex asparagine (N)-linked oligosaccharidesin vertebrates proceeds with the linkage of N-acetylglucosamine(GlcNAc) to the core mannose residues. UDP-N-acetylglucosamine:ß-D-mannosideß1–4 N-acetylglucosaminyltransferase III (GlcNAc-TIII,EC2.4.1.144) catalyzes the addition of GlcNAc to the mannosethat is itself ß1–4 linked to underlying N-acetylglucosamine.GlcNAc-TIII thereby produces what is known as a ‘bisecting’GlcNAc linkage which is found on various hybrid and complexN-glycans. GlcNAc-TIII can also play a regulatory role in N-glycanbiosynthesis as addition of the bisecting GlcNAc eliminatesthe potential for     

Molecular dynamics simulations of high-mannose oligosaccharides

Conformations of several high-mannose-type oligosaccharidesthat are generated during the biosynthetic degradation of Man₉GlcNAc₂to Man₅GlcNAc₂ have been studied by molecular dynamics (MD).Simulations were performed on NCI-FCRDC's Cray Y-MP 8D/8128supercomputer using Biosym's CVFF force field for 1000 Ps withdifferent initial conformations. The conformations of the two1,3- and the two 1,6-linkages in each oligomannose were different,suggesting that deriving oligosaccharide conformations basedon the conformational preferences of the constituent disaccharidefragments will not always yield correct results. Unlike otheroligomannoses, Man₉GlcNAc₂ appears to take more than one distinctconformation around the core 1,6-linkage. These various conformationsmay play an important role in determining the processing pathways.Using the data on the preferred conformations of these oligomannosesand the available experimental results, possible pathways forprocessing Man₉GlcNAc₂ to Man₅GlcNAc₂ by 1,2-linkage-specificmannosidases have been proposed. Conformational analysis ofMan₅GlcNAc₂ indicates that the addition of ß1,2-GlcNActo the 1,3-linked core mannose, besides serving as a prerequisitefor mannosidase II action as suggested earlier, may also preventthe removal of 1,3-mannose. The MD simulations also suggestthat the processing of the precursor oligosaccharide duringAsn-linked complex and hybrid glycan biosynthesis proceeds ina well-defined pathway involving more than one 1,2-linkage-specificmannosidase. Knowledge of the conformation of the processingintermediates obtained from the present study can be used todesign highly specific substrate analogues to inhibit a particularmannosidase, thereby blocking one processing pathway withoutinterfering with the others. carbohydrates conformation glycosidase inhibitors mannosidase oligosaccharide processing     

Terminal {beta}-linked tyvelose creates unique epitopes in Trichinella spiralis glycan antigens

Indirect evidence that the immunodominant N-glycans of the parasite,Trichinella spiralis are capped by novel ß-linked3,6-dideoxy-D-arabinohexopyranosyl residues (tyvelase, Tyv)was obtained from immunochemical assays employing monoclonalantibodies and synthetic oligosaccharides. Three of four previouslycharacterized monoclonal antibodies generated from the lymphocytesof T.spiralis infected rats bind BSA glycoconjugates bearingthe synthetic epitope ß-D-Tyvp(1     

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsinwere analysed by sequential exoglycosidase digestion and gelfiltration chromatography, following reductive tritiation. Inaddition, selected tryptic glycopeptides obtained from frogretinal rod outer segment membranes were examined by electrospraymass spectrometry (ES-MS), fast atom bombardment mass spectrometry(FAB-MS), amino acid sequence and composition analysis, andcarbohydrate composition analysis. The amino acid sequence datademonstrated that the glycopeptides were derived from rhodopsinand confirmed the presence of twoN-glycosylation sites, at residuesAsn² and Asn¹⁵. The predominant glycan (60% of total) had thestructure GlcNAcß1–2Man1–3(Man1–6)Manß1–4GlcNAcß1–4GlcNAc-(Asn),with the remaining structures containing 1–3 additionalhexose residues, as reported previously for bovine rhodopsin.Unlike bovine rhodopsin, however, a sizable fraction of thetotal giycans of frog rhodopsin also contained sialic acid (NeuAc),with the sialylated oligosaccharides being present exclusivelyat the Asn² site. FAB-MS analysis of oligosaccharides releasedfrom the Asn² site gave, among other signals, an abundant quasimolecularion corresponding to a glycan of composition NeuAc₁Hex₆HexNAc₃(where Hex is hexose and HexNAc is N-acetylhexosamine), consistentwith a hybrid structure. The potential biological implicationsof these results are discussed in the context of rod outer segmentmembrane renewal. glycoforms oligosaccharide structure rhodopsin     

Galatosylation and sialylation of mammalian glycoproteins produced by baculovirus-madiated gene expression in insect cells

The baculovirus expression vector system (BEVS) is used extensively for the production of proteins from exogenous cDNAs. However, BEVS is not ideal for pharmaceutical production of glycoproteins owing to the properties of the N-glycans in the expressed products and that insect cells lack several of the enzymes required for mammalian-type N-glycan synthesis. This study describes the effective mammalian-like production of glycoproteins, such as β-1,4-galactosyltransferase and α-2,6-sialyltransferase, in the insect cell line Sf9.Revisions requested 13 April 2005; Revisions received 17 May 2005     

Radioimmunoassay of Gibberellins A5 and A20

Polyclonal anti-GA₅-antiseruin and anti-GA₂₀-antiserum wereprepared by immunizing rabbits with conjugates of N-GA₅-ß-alanylBSA and N-GA₂₀-ß-alanyl BSA. By radioimmunoassay usingthese antisera, GA₅ and GA₂₀ in developing fruits of Pharbitisnil Chois were analyzed after several purification steps andthe results were compared with those obtained by GC-MS to examinethe reliability of the analysis by radioimmunoassay. The analyticalresults by radioimmunoassay did not agree with those by GC-MSuntil two-step purifications by HPLC, indicating that samplesmust be suitably purified prior to radioimmunoassay to obtainreliable results. (Received November 13, 1986; Accepted April 17, 1987)