Glycoconjugate Journal (GLYCOCONJUGATE J)

Publications in this journal

• Article: The glycan moieties of the crustacean egg yolk protein. Does a glucose cap on a secreted protein make sense?

  Ziv Roth, Shmuel Parnes, Simy Wiel, Amir Sagi, Nili Zmora, J. Sook Chung, Isam Khalaia
  Glycoconjugate Journal 02/2013; 27(159):169.
• Article: First synthesis of two deoxy Lewisx pentaosyl glycosphingolipids

  Yun Luo, Chafika Gourmala, Dengxiang Dong, Florent Barbault, Botao Fan, Yongzhou Hu, Yongmin Zhang
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  ABSTRACT: The Lewisx–Lewisx interaction has been increasingly studied, using a variety of techniques including nuclear magnetic resonance spectroscopy, mass spectrometry, vesicle adhesion, atomic force microscopy, and surface plasmon resonance spectroscopy. However, the detailed molecular mechanism of these weak, divalent cation dependent interactions remains unclear, and new models are needed to probe the nature of this phenomenon in term of key roles of the different hydroxyl groups on Lewisx trisaccharide determinant involved in the Lewisx–Lewisx interaction. An interesting solution is to synthesize a series of Lewisx pentaosyl glycosphingolipid derivatives in which one of the eight hydroxyl groups of Lewisx trisaccharide is replaced by a hydrogen atom, and to test the adhesion induced by interaction of these derivatives, in order to gain insight into the functions played by the hydroxyl groups of the Lewisx trisaccharide. This article describes the synthesis of 3d-deoxy and 4d-deoxy Lewisx pentaosyl glycosphingolipids, to be used for study of the Lewisx–Lewisx interaction.
  Glycoconjugate Journal 04/2012; 25(4):335-344.
• Article: Endothelial cell–laminin interaction: modulation of LDH expression involves α6β4 integrin–FAK–p38MAPK pathway

  P. R. Sudhakaran, R. I. Viji, M. S. Kiran, V. B. Sameer Kumar
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  ABSTRACT: One of the possible mechanisms of the angiogenic effect of laminin (Ln) involves modulation of the biological activity of VEGF by regulating poly ADP ribosylation (PAR). PAR modification of VEGF was found to be related with the changes in NAD+ associated with a shift in LDH isoenzymes. Further investigations on LDH gene expression in HUVECs suggested that the effect of Ln was mediated through α6β4 integrin–FAK–src-p38 MAPK pathway. This was evidenced by (a) co-immunoprecipitation of β4 integrin with α6 subunit, (b) activation by tyrosine phosphorylation of β4 integrin and FAK, (c) co-immunoprecipitation of FAK with β4 and with adapter protein, src, (d) increased phosphorylation of p38 MAPK in cells maintained on Ln and (e) blocking of effect of Ln on LDH-B gene expression by inhibition of p38 MAPK. Increase in serine phosphorylation of c-fos and c-jun and higher levels of heterodimers of AP-1 in the nucleus in cells maintained on Ln suggested activation of AP-1 transcription factor. These results provide evidence for modulation of endothelial cell function relevant to angiogenesis by Ln through α6β4 integrin.
  Glycoconjugate Journal 04/2012; 26(6):697-704.
• Article: Molecular characterization of pig α2,3-Gal-β1,3-GalNAc-α2,6-sialyltransferase (pST6GalNAc IV) gene specific for Neu5Acα2-3Galβ1-3GalNAc trisaccharide structure

  Hyun-Kwon Ko, Kwon-Ho Song, Un-Ho Jin, Hwan-Hoo Seong, Young-Chae Chang, Nam-Hyung Kim, Dong-Soo Kim, Young-Choon Lee, Cheorl-Ho Kim
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  ABSTRACT: Sialic acids of glycoconjugates play crucial roles in various biological processes, such as cell-cell communication and cell-substrate interaction. A sisalyltransferase, ST6GalNAc IV (Neu5Ac-α2,3-Gal-β1,3-GalNAc-α2,6-sialyltransferase), catalyzes the formation of α2-6-linkages onto GalNAc residues of O-glycosidically linked Ser/Thr of proteins. In this study, we cloned the pig ST6GalNAc IV (pST6GalNAc IV) and investigated its functional characterization. pST6GalNAc IV cDNA has been isolated from pig liver tissues and it contains an entire open reading frame (ORF, 906bp) coding for 302 amino acid residues. Entire ORF of pST6GalNAc IV containing sialylmotif ‘L’—(Large), ‘S’—(Small) and ‘—VS’ (Very small) has a high degree of sequence similarity with Homo sapiens (90%), Pan troglodytes (91%) and Mus musculus (87%). Expression of pST6GalNAc IV mRNA in various pig tissues was identified by reverse transcription polymerase chain reaction (RT-PCR) analysis. pST6GalNAc IV mRNA was highly expressed in tongue, muscle and heart, whereas it was not expressed in pancreas. For functional characterization of pST6GalNAc IV gene in pig kidney PK15 cells, we have also established pST6GalNAc IV-transfected PK15 cells, which are stably expressing the pST6GalNAc IV gene. The glycosylation pattern of pST6GalNAc IV-transfected PK15 cells was detected by flow cytometry and immunofluorescence analysis with Maackia amurensis agglutinin (MAA), Maackia amurensis hemagglutinin (MAL II), Sambucus nigra agglutinin (SNA) and peanut agglutinin (PNA) lectins. The specific carbohydrate structures of Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc tetrasaccharide or Neu5Acα2-6GalNAc disaccharide recognized by MAL-II and SNA were revealed to be newly synthesized by pST6GalNAc IV. From the results, it was suggested that the pig pST6GalNAc IV gene is capable of synthesizing Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc tetrasaccharide structures on O-glycoproteins. KeywordsNeu5Ac-α2,3-Gal-β1,3-GalNAc-α2,6-sialyltransferase-Neu5Ac-α2,3-Gal-β1,3(Neu5Acα2,6)GalNAc-Tissue expression-MAA-MAL II-SNA-PNA
  Glycoconjugate Journal 04/2012; 27(3):367-374.
• Article: The evolution of galactose α2,3-sialyltransferase: Cionaintestinalis ST3GAL I/II and Takifugu rubripes ST3GAL II sialylate Galβ1,3GalNAc structures on glycoproteins but not glycolipids

  Friederike Lehmann, Sørge Kelm, Frank Dietz, Mark von Itzstein, Joe Tiralongo
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  ABSTRACT: Sialyltransferases are a family of enzymes catalyzing the transfer of sialic acid residues to terminal non-reducing positions of oligosaccharide chains of glycoproteins and glycolipids. Although expression of sialic acid is well documented in animals of the deuterostomian lineage, sialyltransferases have been predominantly described for relatively recent vertebrate lineages such as birds and mammals. This study outlines the characterization of the only sialyltransferase gene found in the tunicate Ciona intestinalis, the first such report of a non-vertebrate deuterostomian sialyltransferase, which has been discussed as a possible orthologue of the common ancestor of galactose α2,3-sialyltransferases. We also report for the first time the characterization of a ST3Gal II gene from the bony fish Takifugu rubripes. We demonstrate that both genes encode functional α2,3-sialyltransferases that are structurally and functionally related to the ST3Gal family of mammalian sialyltransferases. However, characterization of the recombinant, purified forms of both enzymes reveal novel acceptor substrate specificities, with sialylation of the disaccharide Galβ1-3GalNAc and asialofetuin, but not GM1 or GD1b observed. This is in contrast to the mammalian ST3Gal II that predominantly sialylates gangliosides. Taken together the ceramide binding/recognition site previously proposed for the mouse ST3Gal II might represent a unique feature of mammalian ST3Gal II that is missing in the evolutionary more distant fish and tunicate species reported here. This suggests that during the evolution of the ST3Gal II, probably following the separation of the teleosts, a significant shift in substrate specificity enabling the sialylation of gangliosides took place.
  Glycoconjugate Journal 04/2012; 25(4):323-334.
• Source

  Available from: PubMed Central

  Article: Assay and heterologous expression in Pichia pastoris of plant cell wall type-II membrane anchored glycosyltransferases

  Bent Larsen Petersen, Jack Egelund, Iben Damager, Kirsten Faber, Jacob Krüger Jensen, Zhang Yang, Eric Paul Bennett, Henrik Vibe Scheller, Peter Ulvskov
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  ABSTRACT: Two Arabidopsis xylosyltransferases, designated RGXT1 and RGXT2, were recently expressed in Baculovirus transfected insect cells and by use of the free sugar assay shown to catalyse transfer of D-xylose from UDP-α-D-xylose to L-fucose and derivatives hereof. We have now examined expression of RGXT1 and RGXT2 in Pichia pastoris and compared the two expression systems. Pichia transformants, expressing soluble, secreted forms of RGXT1 and RGXT2 with an N- or C-terminal Flag-tag, accumulated recombinant, hyper-glycosylated proteins at levels between 6 and 16mg protein • L-1 in the media fractions. When incubated with 0.5M L-fucose and UDP-D-xylose all four RGXT1 and RGXT2 variants catalyzed transfer of D-xylose onto L-fucose with estimated turnover numbers between 0.15 and 0.3sec-1, thus demonstrating that a free C-terminus is not required for activity. N- and O-glycanase treatment resulted in deglycosylation of all four proteins, and this caused a loss of xylosyltransferase activity for the C-terminally but not the N-terminally Flag-tagged proteins. The RGXT1 and RGXT2 proteins displayed an absolute requirement for Mn2+ and were active over a broad pH range. Simple dialysis of media fractions or purification on phenyl Sepharose columns increased enzyme activities 2-8 fold enabling direct verification of the product formed in crude assay mixtures using electrospray ionization mass spectrometry. Pichia expressed and dialysed RGXT variants yielded activities within the range 0.011 to 0.013U (1U = 1nmol conversion of substrate • min-1 • µl medium-1) similar to those of RGXT1 and RGXT2 expressed in Baculovirus transfected insect Sf9 cells. In summary, the data presented suggest that Pichia is an attractive host candidate for expression of plant glycosyltransferases.
  Glycoconjugate Journal 04/2012; 26(9):1235-1246.
• Article: The expression patterns of β1,4 galactosyltransferase I and V mRNAs, and Galβ1-4GlcNAc group in rat gastrocnemius muscles post sciatic nerve injury

  Meijuan Yan, Chun Cheng, Fei Ding, Jing Jiang, Lingling Gao, Chunlin Xia, Aiguo Shen
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  ABSTRACT: Glycosylation is one of the most important post-translational modifications. It is clear that the single step of β1,4-galactosylation is performed by a family of β1,4-galactosyltransferases (β1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. β1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides and play roles in sciatic nerve regeneration after sciatic nerve injury. In the present study, the expression of β1,4-galactosyltransferase (β1,4-GalT) I, V mRNAs and Galβ1-4GlcNAc group were examined in rat gastrocnemius muscles after sciatic nerve crush and transection. Real time PCR revealed that β1,4-GalT I and V mRNAs expressed at a high level in normal gastrocnemius muscles and decreased gradually from 6h, reached the lowest level at 2weeks, then restored gradually to relatively normal level at 4weeks after sciatic nerve crush. In contrast, in sciatic nerve transection model, β1,4-GalT I and V mRNAs decreased gradually from 6h, and remained on a low level at 4weeks in gastrocnemius muscles after sciatic nerve transection. In situ hybridization indicated that β1,4-GalT I and V mRNAs localized in numerous myocytes and muscle satellite cells under normal conditions and at 4weeks after sciatic nerve crush, and in a few muscle satellite cells at 4weeks after sciatic nerve transection. Furthermore, lectin blotting showed that the expression level of the Galβ1–4GlcNAc group decreased from 6h, reached the lowest level at 2weeks, and restored to relatively normal level at 4weeks after sciatic nerve crush. RCA-I lectin histochemistry demonstrated that Galβ1–4GlcNAc group localized in numerous membranes of myocytes and muscle satellite cells in normal and at 4weeks after sciatic nerve crush, and in a few muscle satellite cells at 2 and 4weeks after sciatic nerve transection. These results indicated that the expressions of β1,4-GalT I, V mRNAs and Galβ1–4GlcNAc group were involved in the process of denervation and reinnervation, which suggests that β1,4-GalT I, V mRNAs and Galβ1-4GlcNAc group may play an important role in the muscle regeneration.
  Glycoconjugate Journal 04/2012; 25(7):685-701.
• Article: UDP-Gal: N-acetylglucosamine β 1–4 galactosyltransferase expressing live attenuated parasites as vaccine for visceral leishmaniasis

  Siddhartha Kumar Bhaumik, Manoj Kumar Singh, Subir Karmakar, Tripti De
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  ABSTRACT: As compared to cutaneous leishmaniasis, vaccination against visceral leishmaniasis (VL) has received limited attention. In this study, we demonstrate for the first time that an UDP-Galactose: N-acetylglucosamine β 1–4 galactosyltransferase (GenBank Accession No. EF159943) expressing attenuated LD clonal population (A-LD) is able to confer protection against the experimental challenge with the virulent LD AG83 parasite. A-LD was also effective in established leishmania infection. The vaccinated animals showed both cell mediated (in vitro T-cell proliferation, and DTH response) and humoral responses (Th1 type). These results demonstrate the potential of the attenuated clones as an immunotherapeutic and immunoprophylactic agent against visceral leishmaniasis.
  Glycoconjugate Journal 04/2012; 26(6):663-673.
• Article: Virulence attenuation of a UDP-galactose/N-acetylglucosamine β1,4 galactosyltransferase expressing Leishmania donovani promastigote

  S. K. Bhaumik, M. Singh, R. Basu, S. Bhaumik, K. Roychoudhury, K. Naskar, S. Roy, T. De
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  ABSTRACT: Protozoan parasites of the genus Leishmania are the causative agent of leishmaniasis, a disease whose manifestations in humans range from mild cutaneous lesions to fatal visceral infections. Human visceral leishmaniasis is caused by Leishmania donovani. Long-term culture in vitro leads to the attenuation of the parasite. This loss of parasite virulence is associated with the expression of a developmentally regulated UDP-Galactose/N-acetylglucosamine β 1–4 galactosyltransferase and galactose terminal glycoconjugates as determined by their agglutination with the pea nut agglutinin (PNA). Thus, all promastigotes passaged for more than 11 times were 100% agglutinated with PNA, and represent a homogeneous population of avirulent parasites. Identical concentrations of PNA failed to agglutinate promastigotes passaged for ≤5 times. These PNA− promastigotes were virulent. Promastigotes passaged from 5 to 10 times showed a mixed population. The identity of populations defined by virulence and PNA agglutination was confirmed by isolating PNA+ avirulent and PNA− virulent clones from the 7th passage promastigotes. Only the PNA+ clones triggered macrophage microbicidal activity. The PNA+ clones lacked lipophosphoglycan. Intravenous administration of [14C] galactose-labeled parasite in BALB/c mice resulted in rapid clearance of the parasite from blood with a concomitant accumulation in the liver. By enzymatic assay and RT-PCR we have shown the association of a UDP-Galactose/N-acetylglucosamine β1,4 galactosyltransferase with only the attenuated clones. By immunofluorescence we demonstrated that the enzyme is located in the Golgi apparatus. By western blot analysis and SDS-PAGE of the affinity-purified protein, we have been able to identify a 29KDa galactose terminal protein from the avirulent clones.
  Glycoconjugate Journal 04/2012; 25(5):459-472.
• Article: Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates

  E.P. Smorodin, O.A. Kurtenkov, B.L. Sergeyev, G.V. Pazynina, N.V. Bovin
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  ABSTRACT: The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)—based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galβ1-3GalNAcα- and Galβ1-3GalNAcβ-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcα-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 × 10−8 M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcβ and GalNAcβ1-3GalNAcβ ligands was found in human serum. Published in 2004.
  Glycoconjugate Journal 04/2012; 20(2):83-89.
• Article: Versatile strategy for the synthesis of biotin-labelled glycans, their immobilization to establish a bioactive surface and interaction studies with a lectin on a biochip

  F. Javier Muñoz, Ángel Rumbero, José V. Sinisterra, J. Ignacio Santos, Sabine André, Hans-J. Gabius, Jesús Jiménez-Barbero, María J. Hernáiz
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  ABSTRACT: The emerging role of glycans as versatile biochemical signals in diverse aspects of cellular sociology calls for establishment of sensitive methods to monitor carbohydrate recognition by receptors such as lectins. Most of these techniques involve the immobilization of one of the binding partners on a surface, e.g. atomic force microscopy, glycan array and Surface Plasmon Resonance (SPR), hereby simulating cell surface presentation. Here, we report the synthesis of fluorescent glycoconjugates, with a functionalization strategy which avoids the frequently occurring ring opening at the reducing end for further immobilization on a surface or derivatization with biotin. In order to improve the versatility of these derivatized glycans for biological studies, a new approach for the synthesis of biotinylated and fluorescent glycans has also been realized. Finally, to illustrate their usefulness the neoglycoconjugates were immobilized on different surfaces, and the interaction analysis with a model lectin, the toxin from mistletoe, proved them to act as potent ligands, underscoring the merit of the presented synthetic approach.
  Glycoconjugate Journal 04/2012; 25(7):633-646.
• Article: Generation of novel chimeric LacdiNAcS by gene fusion of α-lactalbumin and β1,4-galactosyltransferase 1

  Su-Il Do
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  ABSTRACT: Novel chimeric lacdiNAc (GalNAc(β1-4)GlcNAc) synthase (c-LacdiNAcS) was generated by gene fusion of α-lactalbumin (α-LA) and β1,4-galactosyltransferase 1 (β1,4-GalT1). c-LacdiNAcS was expressed in Lec8 Chinese hamster ovary (Lec8 CHO) cells and exhibited N-acetylgalactosaminyltransferase (GalNAcT) activity in the absence of exogenous α-LA as well as other glycosyltransferase activities including lactose synthase (LacS), and β1,4-GalT. These glycosyltransferase activities of c-LacdiNAcS were compared to those activities induced in LacS system under the co-presence of bovine β1,4-GalT1 and α-LA, indicating that each domain of α-LA and β1,4-GalT1 on c-LacdiNAcS is not only folding correctly, but also interacting together. Furthermore, c-LacdiNAcS was found to be auto-lacdiNAcylated and can synthesize lacdiNAc structures on cellular glycoproteins, demonstrating that GalNAcT activity of c-LacdiNAcS is functional in Lec8 CHO cells.
  Glycoconjugate Journal 04/2012; 26(5):567-575.
• Article: Could Nanodiamonds, be a Girl's Best Friends?

  A.H. Siriwardena, Alexandre Barras, Fernando Ariel Martin, Omprakash Bande, Jean-Sébastien Baumann, Jean-Marc Ghigo, Rabah oukherroub, Christophe Beloin, Sabine Szunerits
  Glycoconjugate Journal 01/2011; 28:216.
• Article: N-glycan trimming by glucosidase II is essential for Arabidopsis development.

  Pravina Soussilane, Cecilia D'Alessio, Thomas Paccalet, Anne-Catherine Fitchette, Armando Parodi, Richard Williamson, Carole Plasson, Loïc Faye, Véronique Gomord
  Glycoconjugate Journal 02/2009;
• Article: Obituary: Hal Dixon.

  Hans Vliegenthart
  Glycoconjugate Journal 12/2008;