Occurrence of terminal {alpha}2 ->8-linked disialylated poly-N-acetyllactosamine chains with Lex and I antigenic glycotopes in tetraantennary arms of an N-linked glycoprotein isolated from rainbow trout ovarian fluid

The Pronase digestion of a 54K glycoprotein present in ovarianfluid of rainbow trout yielded a major glycopeptide. Carbohydratecompositional analysis revealed that this glycopeptide was likelyto possess a single large N-glycan chain having low molecularweight oligomers of N-acetylneuraminic acid (oligoNeu5Ac). Structuralstudies of this glycopeptide revealed novel     

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     

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     

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     

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.     

Intracellular proteolytic processing of proopiomelanocortin in heterologous COS-1 cells by the yeast KEX2 endoprotease

We have transiently expressed the yeast KEX2 gene together with the proopiomelanocortin (POMC) cDNA in COS-1 cells. Characterization of the POMC-related immunoreactive peptides by gel permeation and reversed-phase high pressure liquid chromatography showed that the KEX2 enzyme was active and capable of carrying out cleavage of POMC to release the authentic maturation product beta-endorphin(1-31). Peptides resembling beta-lipotropin, the amino terminal glycopeptide, and ACTH(1-39) were also detected as major products in the cell extracts. Our results indicate that the KEX2 enzyme can proteolytically release from POMC a set of peptides similar to that normally found in interior pituitary.     

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     

Calcitonin is not contained within the common precursor to corticotropin and endorphin in the rat.

The suggestion that calcitonin is contained within the structure of the common precursor to ACTH and endorphin was examined. Immunohistochemical staining demonstrated calcitonin in thyroid parafollicular cells, and ACTH and 16K fragment in ACTH/endorphin cells of pituitary. No 16K fragment immunostaining was detected in thyroid parafollicular cells; no calcitonin staining was detected in pituitary. Immunoprecipitation of [³⁵S]methionine-labeled molecules synthesized by rat intermediate pituitary cells demonstrated that neither 30K precursor, 16K fragment nor any other major labeled cell product was recognized by calcitonin antiserum. Analyses of tryptic peptides of 30K precursor indicated that peptides expected from calcitonin were not present in 30K precursor.     

Identification of a Royal Jelly Glycoprotein That Carries Unique Complex-Type N-Glycans Harboring the T-Antigen (Galβ1-3GalNAc) Unit

In this study, we identified a royal jelly glycoprotein (RJG) that carries a unique complex-type N-glycans harboring the T-antigen (Galβ1-3GalNAc) unit. The amino acid sequence of the tryptic glycopeptide harboring the T-antigen unit was G-E-S-L-X-K (X might be glycosylated Asn), confirmed in the major royal jelly glycoprotein 1 (MRJP1), which is also expressed in the mushroom body of the honeybee brain.     

Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin

Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc N-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this N-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications.     

Structural feature of N-glycans of bamboo shoot glycoproteins: useful source of plant antigenic N-glycans

An effective method to prepare plant complex type (PCT) N-glycans in large amounts has been required to evaluate their immunological activity. In this study, we found that glycoproteins in bamboo shoots predominantly carry PCT N-glycans including the Lewis a epitope-containing ones, suggesting that bamboo shoot is an excellent source for the plant antigenic glycans to synthesize immunoactive neoglycopolymers.     

A two-step enzymatic glycosylation of polypeptides with complex N-glycans

A chemoenyzmatic method for direct glycosylation of polypeptides is described. The method consists of two site-specific enzymatic glycosylation steps: introduction of a glucose moiety at the consensus N-glycosylation sequence (NXS/T) in a polypeptide by an N-glycosyltransferase (NGT) and attachment of a complex N-glycan to the glucose primer by an endoglycosidase (ENGase)-catalyzed transglycosylation. Our experiments demonstrated that a relatively small excess of the UDP-Glc (the donor substrate) was sufficient for an effective glucosylation of polypeptides by the NGT, and different high-mannose and complex type N-glycans could be readily transferred to the glucose moiety by ENGases to provide full-size glycopeptides. The usefulness of the chemoenzymatic method was exemplified by an efficient synthesis of a complex glycoform of polypeptide C34, a potent HIV inhibitor derived from HIV-1 gp41. A comparative study indicated that the Glc-peptide was equally efficient as the natural GlcNAc-peptide to serve as an acceptor in the transglycosylation with sugar oxazoline as the donor substrate. Interestingly, the Glc–Asn linked glycopeptide was completely resistant to PNGase F digestion, in contrast to the GlcNAc–Asn linked natural glycopeptide that is an excellent substrate for hydrolysis. In addition, the Glc–Asn linked glycopeptide showed at least 10-fold lower hydrolytic activity toward Endo-M than the natural GlcNAc–Asn linked glycopeptide. The chemoenzymatic glycosylation method described here provides an efficient way to introducing complex N-glycans into polypeptides, for gain of novel properties that could be valuable for drug discovery.     

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     

Solid-Phase Synthesis of Core 8 O-Glycan-Linked MUC5AC Glycopeptide

The benzyl-protected disaccharide building blocks of core 8 O-glycan (15a/15b) for glycopeptide were stereoselectively synthesized by two glycosidation reactions with the glycosyl fluoride method. The building blocks were utilized in the solid-phase synthesis of a glycopeptide carrying two O-glycans with the consensus sequence of the tandem-repeat domain of MUC5AC. The synthetic glycopeptide was detached from the resin with reagent K, and subsequent debenzylation under conditions of low-acidity TfOH afforded glycopeptide 2. The synthetic sample will be used as a suitable standard in studies of the physicochemical or immunochemical characterization of mucin glycoforms.     

Glycoform Analysis of N-Glycans Linked to Glycoproteins Expressed in Rice Culture Cells: Predominant Occurrence of Complex Type N-Glycans

Although it has been found that plant endo-β-N-acetylglucosaminidase shows strong activity towards denatured glycoproteins and glycopeptides with high-mannose type N-glycans and free high-mannose type N-glycans bearing the chitobiosyl unit, the endogenous substrates for plant endoglycosidase have not yet been identified. Recently we purified and characterized an endo-β-N-acetylglucosaminidase from rice culture cells and identified the gene encoded (Maeda, M., and Kimura, Y., Trends Glycosci. Glycotech., 17, 205–214 (2005)). Furthermore, we found structural features of free N-glycans in the cells, indicating that high-mannose type species (Man_9-5GlcNAc₁) occur at concentration of several micromolar (μM). Hence, in this study we analyzed glycoform of N-glycans linked to glycoproteins expressed in rice culture cells to see whether endogenous glycoproteinous substrate occurs in reasonable amounts. Structural analysis revealed that more than 95% of total N-glycans linked to glycoproteins in the rice cells had the plant complex type structure, including Lewis a epitope-harboring type, although high-mannose type structures account for less than 5% of total N-glycans.     

Rice α-fucosidase active against plant complex type N-glycans containing Lewis a epitope: purification and characterization

Rice α-fucosidase (α-fucosidase Os, 58 kDa) that is active for α1-4 fucosyl linkage in Lewis a unit of plant N-glycans was purified to homogeneity. α-fucosidase Os showed activity against α1-3 fucosyl linkage in Lacto-N-fucopentaose III but not α1-3 fucosyl linkage in the core of plant N-glycans. The N-terminal sequence of α-fucosidase Os was identified as A-A-P-T-P-P-P-L-, and this sequence was found in the amino acid sequence of the putative rice α-fucosidase 1 (Os04g0560400).     

Monoclonal antibody LU-BCRU-G7 against a breast tumour-associated glycoprotein recognizes the disaccharide Gal{beta}1-3GlcNAc

The monoclonal antibody LU-BCRU-G7, that was generated by invitro immunization, shows clinical value as a prognostic markerin early stage breast carcinoma. It has now been characterizedwith regard to its binding epitope. Using a recently describedmethod based on the construction of N-substituted polyacrylamide(PAA) derivatives of carbohydrates (pseudopolysaccharides),the structure of the epitope for the monoclonal antibody LU-BCRU-G7has been determined. Competitive binding assays and inhibitoryenzyme-linked immunosorbent assays (ELISAs) using these pseudopolysaccharideshave shown the LU-BCRU-G7 epitope to be a disaccharide Galß1-3GlcNAc(Le^c; where Gal is D-galactose, Glc is D-glucose and GlcNAcis N-acetyl-D-glucosainine). Both galactose and N-acetyl glucosaminemoieties are essential for binding. Substitution on C-2 or C-3of the terminal galactose abolished binding, as did galactose-terminated oligosaccharides. The galactose moiety alone, asexpressed by the Galß-PAA conjugate, appeared to hea more important feature of the epitope than the GlcNAc-PAAconjugate, which failed to bind or inhibit the LU-BCRU-G7 antibody.In the N-acetyl glucosamine moiety, binding was decreased butnot eliminated by fucose substitution, as in Le^a, or changein configuration of C-4, as in Galß1-3GlcNAc. Omissionof the NAc group resulted in complete loss of activity. Thetetrasaccharide lacto-N-tetraose, although containing the terminalLe^c disaccharide, does not react with the antibody, suggestingconformational interference of the binding site. These findingsshow that the monoclonal antibody LU-BCRU-G7 recognizes a terminalisolactosamine fragment on a tumour-associated glycoprotein,which we have previously shown to be inversely related to survivalin breast cancer. breast cancer Galß1-3GlcNAc LU-BCRU-G7 monoclonal antibody pseudopolysaccharides     

First evidence of human meconium glycoasparagines

During a systematic study of carbohydrate material present inhuman meconium, in addition to the previously described mucins,glycolipids and free oligosaccharides, we have now characterizeda significant quantity of free glycoasparagines. These glycoasparagineshave been isolated from human meconium by a combination of ion-exchange,concanavalin A (ConA)-affinity and high-performance liquid (HPLC)chromatographies. Their structures have been established by400 MHz ¹H-NMR spectroscopy. These compounds are related toN-acetyllactosaminic type structures and are based on the commoncore These glycoasparagines are probably derived from both proteaseand partial exoglycosidase hydrolysis of fetal gastrointestinalN-glycosyl proteins. Their structures are discussed in the contextof the known catabolic pathways of N-glycans glycoasparagine N-glycosyl protein catabolism meconium NMR     

The effect of pepstatin A, an inhibitor of the pro-opiomelanocortin (POMC)-converting enzyme, on POMC processing in mouse intermediate pituitary

In our previous studies, we have purified a unique, paired basic residue-specific, prohormone-converting enzyme from pituitary intermediate lobe secretory vesicles. This enzyme, an aspartyl protease, was shown to cleave the intermediate lobe prohormone, pro-opiomelanocortin (POMC), to adrenocorticotropin, beta-endorphin and a 16 kDa NH2-terminal glycopeptide, in vitro [(1985) J. Biol. Chem. 260, 7194-7205]. To provide some evidence that this enzyme plays a role in prohormone conversion in the intact cell, the ability of pepstatin A, an aspartyl protease inhibitor, to block POMC processing in the mouse intermediate pituitary was investigated. By the use of a radioactive pulse-chase paradigm, [3H]POMC processing was found to be inhibited by 36.4% in pepstatin A-treated intermediate lobes. This result is consistent with the inactivation of pro-opiomelanocortin-converting enzyme by pepstatin A in the intact pituitary and further supports a role of this enzyme in POMC processing in vivo.