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     

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     

Glycosylation Patterns of HIV-1 gp120 Depend on the Type of Expressing Cells and Affect Antibody Recognition

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the interaction between a variably glycosylated envelope glycoprotein (gp120) and host-cell receptors. Approximately half of the molecular mass of gp120 is contributed by N-glycans, which serve as potential epitopes and may shield gp120 from immune recognition. The role of gp120 glycans in the host immune response to HIV-1 has not been comprehensively studied at the molecular level. We developed a new approach to characterize cell-specific gp120 glycosylation, the regulation of glycosylation, and the effect of variable glycosylation on antibody reactivity. A model oligomeric gp120 was expressed in different cell types, including cell lines that represent host-infected cells or cells used to produce gp120 for vaccination purposes. N-Glycosylation of gp120 varied, depending on the cell type used for its expression and the metabolic manipulation during expression. The resultant glycosylation included changes in the ratio of high-mannose to complex N-glycans, terminal decoration, and branching. Differential glycosylation of gp120 affected envelope recognition by polyclonal antibodies from the sera of HIV-1-infected subjects. These results indicate that gp120 glycans contribute to antibody reactivity and should be considered in HIV-1 vaccine design.     

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     

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.     

HL 60 leukaemia cells chemically induced to differentiate retain some surface glycan features of undifferentiated cells not found in normal leukocytes

Human HL 60 myeloid leukaemia ells have the potential to differentiateinto either macrophage-like cells or granulocyte-like cellsunder the stimulus of chemical treatments. Using glycotechnologyprocedures, the glycosylation patterns of differentiated andundifferentiated HL 60 cells were analysed and compared withthose of normal human peripheral monocytes. Both in vitro differentiationsresult in significant morphologic and functional changes, butwe observed that the glycosylation patterns of undifferentiatedand differentiated HL 60 cells exhibit several common glycosidicfeatures that are absent in normal peripheral monocytes: thepresence of (i) bisecting ß-N-acetylglucosamine attachedat the C-4 position of the ß-mannose of polyantennarycomplex-type carbohydrate chains and (ii) complex-type carbohydratechains enriched with non-reducing terminal ß-N-acetylglucosamineresidues. Moreover, the three populations of HL 60 cells expresssmall amounts of biantemary complex-type structures (<6%),whereas normal peripheral monocytes expressed >20% of suchstructures. Thus, the cell glycosylation pattern could reflectthe pathological state of the HL 60 cells. differentiation glycosylation HL 60 cell monocytes     

The Metabolism of Abscisic Acid

The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-¹⁴C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-¹⁴D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-¹⁴C]trans-abscisic acid in an equimolar mixturewith (±)-[2-¹⁴C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-¹⁴C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-¹⁴C]-, and (–)-[2-¹⁴C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-^l4C]ABA.     

Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human {beta}1,2-N-acetylglucosaminyltransferase I

Spodoptera frugiperda (Sf9)-cells differ markedly in their proteinglycosylation capacities from vertebrate cells in that theyare not able to generate complex type oligosaccharide side chains.In order to improve the oligosaccha ride processing propertiesof these cells we have used baculovirus vectors for expressionof human (ß1,2-N-acetylglucosaminyltransferase I (hGNT-I),the enzyme catalysing the crucial step in the pathway leadingto complex type N-glycans in vertebrate cells. One vector (Bac/GNT)was designed to express unmodified GNT-I protein, the secondvector (Bac/tagGNT) to express GNT-I protein with a tag epitopefused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virusa protein of about 50 kDa representing hGNT-I was detected withan antiserum directed against the tag epitope. HGNT-I activitywas increased at least threefold in lysates of infected cellswhen N-acetylglucosamine (GlcNAc)-free ovalbumine was used assubstrate. To monitor hGNT-I activity in intact Sf9-cells, theglycosylation of coexpressed fowl plague virus hemagglutinin(HA) was investigated employing a galactosylation assay andchromatographic analysis of isolated HA N-glycans. Coexpressionof hGNT-I resulted in an at least fourfold increase of HA carryingterminal GlcNAc-residues. The only structure detectable in thisfraction was GlcNAcMan₃GlcNAc₂. These results show that hGNT-Iis functionally active in Sf9-cells and that the N-glycans ofproteins expressed in the baculovirus/insect cell system areelongated by coexpression of glycosyltransferases of vertebrateorigin. Complete complex type oligosaccharide side chains werenot observed when hGNT-I was overexpressed, thus supportingthe concept that Sf9-cells do not contain glycosyltransferasesacting after hGNT-I. ß1,2-N-acetylglucosaminyltransferase I baculovirus expression of recombinant protiens N-glycosylation in Sf9-cells     

Relationship between Flower Induction by Benzoic Acid and its Metabolism in Lemna paucicostata 151

The flower-inducing activities in Lemna paucicostata 151 offour major metabolites of benzoic acid (N-benzoyl aspartate,benzyl 6-O-ß-D-apiofuranosyl-O-ß-D-glucopyranoside,O-benzoyl isocitrate and O-benzoyl malate) were measured, andthe effects on the uptake and metabolism of benzoic acid dueto change in the level of the benzoic acid concentration orto the addition of plant hormones were investigated. N-Benzoylaspartate had weak activity, and O-benzoyl isocitrate and malatehad fairly strong activities, while benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranosideshowed no activity. As the concentration of benzoic acid rose,the ratio of N-benzoyl aspartate increased and that of benzyl6-O-ß-D-apiofuranosyl-O-ß-D-glucopyranosidedecreased. GA₃ and IAA, inhibitors of flower induction by benzoicacid, seemed to promote conversion to N-benzoyl aspartate insteadof to benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranoside.The conversion to N-benzoyl aspartate was considered to be adetoxification process and that to benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranosidemay be directly related to flower induction in Lemna. (Received November 2, 1987; Accepted January 23, 1988)     

Site-specific glycan analysis of human chorionic gonadotropin beta-subunit from malignancies and pregnancy by liquid chromatography--electrospray mass spectrometry

Glycosylation is an important posttranslational modificationin proteins, and aberrant glycosylation occurs in malignancies.Human chorionic gonadotropin (hCG) is a glycoprotein hormoneproduced in high concentrations during pregnancy. It is alsoexpressed as particular glycoforms by certain malignancies.These glycoforms, which are called "hyperglycosylated" hCG (hCGh),have been reported to contain more complex glycan moieties.We have analyzed tryptic glycopeptides of the ß-subunitof hCG of various origins by liquid chromatography (LC) connectedto an electrospray mass spectrometer. Site-specific glycan structureswere visualized by the use of differential expression analysissoftware. hCGß was purified from urine of two patientswith testicular cancer, one with choriocarcinoma, one with aninvasive mole, two pregnant women at early and late gestation,from a pharmaceutical preparation and culture medium of a choriocarcinomacell line. N-glycans at Asn-13 and Asn-30 as well as O-glycansat Ser-121, Ser-127, Ser-132, and Ser-138 were characterized.In all samples, the major type of N-glycan was a biantennarycomplex-type structure, but triantennary structures linked toAsn-30 as well as fucosylation of the Asn-13-bound glycan areincreased in cancer-derived hCGß. There were significantsite-specific differences in the O-glycans, with constant core-2glycans at Ser-121, core-1 glycans at Ser-138, and putativesites unoccupied by any glycan. Core-2 glycans at either Ser-127or Ser-132 were enriched in cancer. The glycans of free hCGßwere larger and had a higher fucose content of Asn-13-linkedoligosaccharides than intact hCG. This may facilitate the detectionof this malignancy-associated variant by a lectin assay. Analysisof hCGh affinity purified with antibody B152 confirmed thatthis antibody recognizes a core-2 glycan on Ser-132.     

Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission

The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1_NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.     

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     

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine     

Microheterogeneity of N-glycosylation on a stylar self-incompatibility glycoprotein of Nicotiana alata

Gametophytic self-incompatibility, a mechanism that preventsinbreeding in some families of flowering plants, is mediatedby the products of a single genetic locus, the S-locus. Theproducts of the S-gene in the female sexual tissues of Nicotianaalata are an allelic series of glycoproteins with RNase activity.In this study, we report on the microheterogeneity of N-linkedglycosylation at the four potential N-glycosylation sites ofthe S²-glycoprotein. The S-glycoproteins from N.alata containfrom one to five potential N-glycosylation sites based on theconsensus sequence Asn-Xaa-Ser/Thr. The S²-glycoprotein containsfour potential N-glycosylation sites at Asn²⁷, Asn³⁷, Asn¹³⁸and Asn¹⁵⁰, designated sites I, n, IV and V, respectively. SiteIII is absent from the S₂-glycoprotein. Analysis of glycopeptidesgenerated from the S₂-glycoprotein by trypsin and chymotrypsindigestions revealed the types of glycans and the degree of microheterogeneitypresent at each site. Sites I (Asn²⁷) and IV (Asn¹³⁸) displaymicroheterogeneity, site II (Asn³⁷) contains only a single typeof N-glycan, and site V (Asn¹⁵⁰) is not glycosylated. The microheterogeneityobserved at site I on the S₂-glycoprotein is the same as thatobserved at the only site, site I, on the Srglycoprotein (Woodwardet al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylationconsensus sequence at site I is conserved in all S-glycoproteinsfrom other species of self-incompatible solanaceous plants,glycosylation at this site may be important to their function.No other post-translational modifications (e.g. O-glycosylation,phosphorylation) were detected on the S₂-glycoprotein. fertilization microheterogeneity N-glycans plants RNase     

Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)_n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis^x and sialyl-Lewis^x determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.     

Characterization of serum {beta}-glucuronyltransferase involved in chondroitin sulfate biosynthesis

We studied a glucuronyltransferase involved in chondroitin sulfate(CS) biosynthesis in a preparation obtained from fetal bovineserum by heparin-Sepharose affinity chromatography. This enzymetransferred GlcA from UDP-GlcA to the nonreducing GalNAc residuesof polymeric chondroitin. It required Mn²⁺ for maximal activityand showed a sharp pH optimum between pH 5.5 and 6.0. The apparentKm value of the glucuronyltransferase for UDP-GlcA was 51 µM.The specificity was investigated using structurally definedacceptor substrates, which consisted of chemically synthesizedtri-, penta-, and heptasaccharide-serines and various odd-numberedoligosaccharides with a GalNAc residue at the nonreducing terminus,prepared from chondroitin and CS by chondroitinase ABC digestionfollowed by mercuric acetate treatment. The enzyme utilizeda heptasaccharide-serine GalNAcß1-4GlcAß1-3GalNAcß1-4GlcAß1-3Galß1-3Galß1-4Xylß1-O-Serand a pentasaccharide-serine GalNAcß 4GlcAß1-3Galß1-3Galß1-4Xylß1-O-Seras acceptors. In contrast, neither a trisaccharide-serine Galß1-3Galß1-4Xylß1-O-Sernor an     

O-Linked fucose in glycoproteins from Chinese hamster ovary cells

We report our discovery that many glycoproteins synthesizedby Chinese hamster ovary (CHO) cells contain fucose in O-glycosidiclinkage to polypeptide. To enrich for the possible presenceof O-linked fucose, we studied the lectin-resistant mutant ofCHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferaseI and are therefore unable to synthesize complex-type N-linkedoligosaccharides. Lec1 cells were metabolically radiolabelledwith [6-³H]fucose and total glycoproteins were isolated. Glycopeptideswere prepared by proteolysis and fractionated by chromatographyon a column of concanavalin A (Con A)— Sepharose. Thesets of fractionated glycopeptides were treated with mild base/borohydrideto effect the ß-elimination reaction and release potentialO-linked fucosyl residues. The ß-elimination produced[³H]fucitol quantitatively from [³H]fucose-labelled glycopeptidesnot bound by Con A-Sepharose, whereas none was generated bytreatment of glycopeptides bound by the lectin. The total [³H]fucose-labelledglycoproteins from Lec1 cells were separated by SDS—PAGEand detected by fluorography. Treatment of selected bands ofdetectable glycoproteins with mild base/borohydride quantitativelygenerated [³H]fucitol. Pretreatment of the glycoproteins withN-glycanase prior to the SDS—PAGE method of analysis causedan enrichment in the percentage of radioactivity recovered as[³H]fucitol. Trypsin treatment of [³H]fucose-labelled intactCHO cells released glycopeptides that contained O-linked fucose,indicating that it is present in surface glycoproteins. Thesefindings demonstrate that many glycoproteins from CHO cellscontain O-linked fucosyl residues and raise new questions aboutits biosynthesis and possible function. fucose glycoproteins monosaccharide O-linked     

Effects of Deoxygibberellin C (DGC) and 16-Deoxo-DGC on Gibberellin 3{beta}-Hydroxylase and Plant Growth

Deoxygibberellin C (DGC), a C/D ring-rearranged isomer of GA₂₀,was shown to inhibit the conversion of [2,3-³H₂]GA₉ to [2-³H]GA₄by gibberellin 3ß-hydroxylase from immature seedsof Phaseolus vulgahs. Deoxygibberellin C inhibited the promotionof growth by exogenously applied GA₂₀ of rice (Oryza sativaL.) seedlings. Evidence is also presented that DGC is a competitiveinhibitor of the 3ß-hydroxylase from P. vulgaris.However, DGC only weakly inhibited the conversion catalyzedby the 3ß-hydroxylase from Cucurbita maxima at highconcentrations, and it did not inhibit the promotion of growthby exogenously applied GA₉ of cucumber (Cucumis sativus) seedlings.These results suggest that the 3ß-hydroxylases fromP. vulgaris and C. maxima have different structural requirementswith respect to their substrates. 16-Deoxo-DGC also inhibitedcatalysis of the same conversion by 3ß-hydroxylasefrom P. vulgaris, and it slightly inhibited the conversion catalyzedby the enzyme from C. maxima. Application of 16-deoxo-DGC causedthe promotion of the growth of seedlings of both rice and cucumber. ³ Present address: Genetic Engineering Center, Korea Instituteof Science and Technology, Daejeon 305–606, Korea ⁴ Present address: Department of Agricultural Chemistry, UtsunomiyaUniversity, Utsunomiya-shi, Tochigi, 321 Japan (Received September 25, 1990; Accepted December 17, 1990)