Showing papers in "Glycobiology in 1995"
TL;DR: It is concluded that glycosylation of the antibody may be important in the clinical outcome of therapy.
Abstract: CAMPATH-1H (where CAMPATH is a trade mark of Wellcome group companies), a humanized IgG antibody used in the therapy of lymphoma, leukaemia and rheumatoid arthritis, has been expressed in Chinese hamster ovary, Y0 myeloma and NS0 myeloma cell lines. These engineered cell lines were grown under different culture conditions, and the antibody isolated and purified. N-Linked oligosaccharides, on the CH2 heavy chain region of the antibody, were isolated and analysed by hydrazinolysis, high-performance anion-exchange chromatography with pulsed amperometric detection, laser-desorption mass spectrometry and sequential exoglycosidase treatment. Both the glycosylation pattern and the biological activity of CAMPATH-1H, as measured by antibody-dependent cell-mediated cytotoxicity, were markedly affected by the cell line used to express the antibody. It is concluded that glycosylation of the antibody may be important in the clinical outcome of therapy.
323 citations
TL;DR: The authors have isolated human genomic DNA clones encompassing 30 kbp of the histo-blood group ABO locus and the locations of the exons have been mapped and the nucleotide sequences of theExon-intron boundaries have been determined.
Abstract: We have isolated human genomic DNA clones encompassing 30 kbp of the histo-blood group ABO locus. The locations of the exons have been mapped and the nucleotide sequences of the exon-intron boundaries have been determined. The human ABO genes consist of at least seven exons, and the coding sequence in the seven coding exons spans over 18 kb of the genomic DNA. The exons range in size from 28 to 688 bp, with most of the coding sequence lying in exon 7.
212 citations
TL;DR: The present study describes the isolation and structural elucidation of seven new oligosaccharides: an octasaccharide, two decasac charides, two dodecasACcharides and two tetradecasacchariding.
Abstract: Porcine mucosal heparin was partially depolymerized with heparin lyase I and then fractionated into low-molecular-weight ( 5000) oligosaccharides by pressure filtration. The high-molecular-weight oligosaccharide mixture (approximately 50 wt% of the starting heparin) also contained intact heparin. This intact polymer complicates oligosaccharide purification. Thus, the low-molecular-weight fraction was used to prepare homogeneous oligosaccharides for structural characterization. The low-molecular-weight oligosaccharide mixture was first fractionated by low-pressure gel permeation chromatography into size-uniform mixtures of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharides and higher oligosaccharides. Each size-fractionated mixture was then purified on the basis of charge by repetitive semi-preparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 14 homogeneous oligosaccharides from disaccharide to tetradecasaccharide. The purity of these heparin-derived oligosaccharides was determined by gradient polyacrylamide gel electrophoresis, analytical strong-anion-exchange high-performance liquid chromatography, capillary electrophoresis and one-dimensional nuclear resonance spectroscopy. The structure of these oligosaccharides was established using 600 MHz two-dimensional nuclear resonance spectroscopy. The spectral methods used included homonuclear correlation spectroscopy, nuclear Overhauser effect spectroscopy and heteronuclear multiple quantum coherence spectroscopy. The 1H/1H connectivities of the protons of each sugar residue in an oligosaccharide were established by two-dimensional homonuclear correlation spectroscopy, while 1H/13C assignments were made using 1H inverse detection. One- and two-dimensional nuclear resonance spectroscopic analysis of these heparin oligosaccharides showed two closely related groups of heparin-oligosaccharides are afforded by enzymatic depolymerization of heparin. One group is fully sulphated, having the structures delta UAp2S(1[-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S( 1]n-->4)-alpha- D-GlcNpS6S, where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid, GlcNp is 2-deoxy-2-aminoglucopyranose, IdoAp is idopyranosyluronic acid, S is sulphate and n = 0-6. The other group of oligosaccharides differ in that they contain beta-D-glucuronic acid in place of the alpha-L-iduronic acid residue nearest to the reducing end. The present study describes the isolation and structural elucidation of seven new oligosaccharides: an octasaccharide, two decasaccharides, two dodecasaccharides and two tetradecasaccharides. The utility of two-dimensional nuclear resonance spectroscopy to determine the structure of complex heparin oligosaccharides is also illustrated.
197 citations
189 citations
TL;DR: The metabolic pathways involved in the attachment to or removal of galactofuranose from glycoconjugates have not yet been elucidated and this is an area of incipient research, but of growing importance, since it will foster the design of inhibitors which may prove to be useful for the treatment of disease.
Abstract: Galactofuranose has been characterized in glycoinositolphospholipid (GIPL) anchor-like structures having a glycerolipid or a ceramide, as in lipopeptidophosphoglycan (LPPG) of Trypanosoma cruzi, in the oligosaccharide core of lipopeptidophosphoglycan (LPG) of Leishmania species, and also modifying high-mannose chains of trypanosomatid glycoproteins. Galactofuranose is usually present linked beta 1-->3 to Man, either as a terminal non-reducing unit, like in LPPG, or in the middle of the oligosaccharide core, as in LPG. The presence in protozoan parasites of galactose in the furanose configuration is a feature which deserves further attention since the mammalian hosts do not appear to produce glycoconjugates containing this structural unit. For that reason, hosts produce antibodies against galactofuranose, which may turn out to be important in understanding the pathogenesis and in the development of diagnostic methods. The metabolic pathways involved in the attachment to or removal of galactofuranose from glycoconjugates have not yet been elucidated. This is an area of incipient research, but of growing importance, since it will foster the design of inhibitors which may prove to be useful for the treatment of disease.
178 citations
171 citations
TL;DR: This work re-examined the reported multiacylated nature of PIMs in order to gain a better insight into their possible roles as biosynthetic precursors of LM and LAM, and presents evidence for triacylated mannophosphoinositide as a common lipid anchor for both LM andLAM.
Abstract: Based on chemical analysis, we have previously concluded that the biologically important lipoarabinomannan (LAM) and lipomannan (LM) from Mycobacterium are multiglycosylated forms of the phosphatidylinositol mannosides (PIMs), the characteristic cell envelope mannophosphoinositides of mycobacteria. Using definitive analytical techniques, we have now re-examined the reported multiacylated nature of PIMs in order to gain a better insight into their possible roles as biosynthetic precursors of LM and LAM. High-sensitivity fast atom bombardment-mass spectrometry analyses of the perdeuteroacetyl and permethyl derivatives of PIMs from Mycobacterium tuberculosis and Mycobacterium leprae enabled us to define the exact fatty acyl compositions of the multiacylated, heterogeneous PIM families, notably the dimannoside (PIM2) and the hexamannoside (PIM6). Specifically, in conjunction with other chemical and gas chromatography-mass spectrometry (GC-MS) analyses, the additional C16 fatty acyl substituent on PIM2 and its lyso form were defined as attached to the C6 position of mannose. We also present evidence for triacylated mannophosphoinositide as a common lipid anchor for both LM and LAM, and further postulate that acylation of PIM2 may constitute a key regulatory step in their biosynthesis.
130 citations
TL;DR: Results indicate that tissue fibronectin, as well as laminin, serve as endogenous ligands for galectin-1, suggesting that galectIn-1 may play a role in assembly of the extracellular matrix, or in the control of cell adhesion based on lectin-extracellular Matrix interaction.
Abstract: A 14K beta-galactoside-binding lectin (galectin-1) is present in many animal tissues. In a search for endogenous ligands, we surveyed galectin-1-binding proteins in human placenta. Extract of human placenta with 2 M urea was applied to a Sepharose 4B column conjugated with galectin-1 purified from frog (Rana catesbeiana) eggs. Two major proteins eluted with 100 mM lactose from the column-bound fraction showed apparent molecular masses of 220 and 180 kDa on SDS-PAGE under reducing conditions. Western blotting analysis using monoclonal antibodies indicated that these proteins were fibronectin and laminin, respectively. Most placental and amniotic fibronectins bound strongly to the column, whereas almost all plasma fibronectin passed through the column. The galectin-1, fibronectin and laminin were immunohistochemically shown to be co-localized in the extracellular matrix of placental tissue. In a cell attachment assay, rhabdosarcoma cells adhered to a plate coated with placental fibronectin, even in the presence of GRGDS peptide, if galectin-1 were also present. This adhesive effect of galectin-1 was inhibited by lactose. These results indicate that tissue fibronectin, as well as laminin, serve as endogenous ligands for galectin-1, suggesting that galectin-1 may play a role in assembly of the extracellular matrix, or in the control of cell adhesion based on lectin-extracellular matrix interaction.
113 citations
TL;DR: Recent studies on the structures and specific functions of different glycans of natural and recombinant glycoprotein hormones have provided valuable insight into the glycobiology of these hormones, which will be useful in the development of diagnostic and therapeutic applications of the glycop protein hormones.
Abstract: Chorionic gonadotrophin and pituitary luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone comprise the family of glycoprotein hormones, which regulate major metabolic and reproductive functions of the body. These are heterodimeric glycoproteins composed of a common alpha subunit and a hormone-specific beta subunit. The N-linked oligosaccharides of these hormones are necessary for proper folding, assembly, secretion, metabolic clearance and biological activity. The free alpha subunit, which is shown to have a physiological function, is maintained in the uncombined form due to its glycan structures. The N-glycans of the glycoprotein hormones contain a variety of terminal residues, which are responsible for the differential targeting and clearance of the hormones. Glycosylation of these hormones is regulated by a variety of physiological and pathological conditions, leading to subtle alterations in their bioactivities. Recent studies on the structures and specific functions of different glycans of natural and recombinant glycoprotein hormones have provided valuable insight into the glycobiology of these hormones. This information will be useful in the development of diagnostic and therapeutic applications of the glycoprotein hormones.
97 citations
TL;DR: This work has developed a method to identify glycosidases with novel specificities using fluorescently labelled carbohydrate substrates and TLC, which led to the discovery that bacteria of the genus Xanthomonas are a rich source of exoglycosidase.
Abstract: Enzymatic analysis of oligosaccharides using exoglycosidases has become a powerful tool for determining the sequence and structure of sugar chains. The principal limitation to these methods has been the lack of highly purified and well-characterized enzymes. Using fluorescently labelled carbohydrate substrates and TLC, we have developed a method to identify glycosidases with novel specificities. This screening method led to the discovery that bacteria of the genus Xanthomonas are a rich source of exoglycosidases. From Xanthomonas manihotis, eight novel exoglycosidases have been isolated and characterized. A novel beta-N-acetylglucosaminidase has been purified that, unlike those previously described, will cleave N-acetylglucosamine without cleaving N-acetylgalactosamine residues. A novel beta-galactosidase has been isolated that preferentially hydrolyses beta(1-->3) galactosyl linkages. Three alpha-mannosidases have been isolated that serve as useful reagents in the analysis of high-mannose oligosaccharide structures: alpha 1-3,6 mannosidase, alpha 1-6 mannosidase and alpha 1-2,3 mannosidase. An alpha 1-3,6 galactosidase has been purified that does not hydrolyse terminal alpha 1-4 galactose residues. Two fucosidases, alpha 1-3,4 fucosidase and alpha 1-2 fucosidase, are similar to enzymes purified from other sources. Together, these glycosidases provide powerful reagents for determining the sequence of complex carbohydrates. Equally important is their usefulness in selectively removing specific sugar residues and thereby creating novel carbohydrates for analysing the biological roles of oligosaccharides.
94 citations
TL;DR: This article is an attempt to provide, in an easily accessible format, a compilation of genes and cDNAs that have been sequenced and deposited in GenBank that encode transferase enzymes involved in eukaryotic glycoprotein or glycolipid biosynthesis.
Abstract: The rapidity with which molecular sequence data are gathered continues to grow. The result is that, for many workers, it is increasingly difficult to keep abreast of the current state of play of molecular cloning, even for those genes that encode proteins of special interest. The clear success of the various worldwide genome projects has made this even more apparent, and by the end of 1996 the complete determination of the nucleotide sequences of the genomes of two eukaryotes, Saccharomyces cerevisiae and Caenorhabditis elegans, will have either been completed or will be nearing completion. This article is an attempt to provide, in an easily accessible format, a compilation of genes and cDNAs that have been sequenced and deposited in GenBank that encode transferase enzymes involved in eukaryotic glycoprotein or glycolipid biosynthesis. The full sequence information can be easily retrieved from a databank, e.g. GenBank, using the relevant accession number(s).
TL;DR: Viridans streptococci that participate in the microbial colonization of teeth have cell wall polysaccharides composed of linear phosphodiester-linked hexa- or heptasaccharide repeating units, each containing a host-like disaccharides motif, either Gal beta 1-->3GalNAc or GalNAc beta 1)--3Gal.
Abstract: Viridans streptococci that participate in the microbial colonization of teeth have cell wall polysaccharides composed of linear phosphodiester-linked hexa- or heptasaccharide repeating units, each containing a host-like disaccharide motif, either Gal beta 1-->3GalNAc or GalNAc beta 1-->3Gal. Whereas strains with GalNAc beta 1-->3Gal-containing polysaccharides co-aggregated with streptococci that possess GalNAc-sensitive lectins, strains with either host-like motif co-aggregated with Actinomyces spp. The latter interactions reflected the specificity of Actinomyces spp. lectins for common features of Gal beta 1-->3GalNAc and GalNAc beta 1-->3Gal. Thus, alpha-linked glycosides of both disaccharides were much more potent inhibitors of co-aggregation than Gal or GalNAc. Six non-bacterial lectins also reacted with the streptococcal polysaccharides. In general, precipitation of each lectin with each polysaccharide involved binding of Gal or GalNAc within the host-like motifs, but not saccharides outside these regions. The lectins of Ricinus communis, Abrus precatorius, Codium fragile and Agaricus bisporus were most reactive with the Gal beta 1-->3GalNAc-containing polysaccharides, the Wisteria floribunda lectin with the GalNAc beta 1-->3Gal-containing polysaccharides and the Bauhinia purpurea lectin with polysaccharides containing either disaccharide. Thus, lectin recognition of the streptococcal cell wall polysaccharides involved either the common or specific sides of the Gal beta 1-->3GalNAc and GalNAc beta 1-->3Gal motifs present within these molecules.
TL;DR: The binding of the lymphocyte membrane L-selectin to endothelial glycosaminoglycans may provide a link between the selectin-mediated and integrin-mediated adhesion systems in leukocyte extravasation cascades.
Abstract: This communication is concerned with the binding specificity of the leukocyte-adhesion molecule L-selectin (leukocyte homing receptor) towards structurally defined sulphated oligosaccharides of the blood group Le(a) and Le(x) series, and of the glycosaminoglycan series heparin, chondroitin sulphate and keratan sulphate. The recombinant soluble form of the rat L-selectin (L-selectin-IgG Fc chimera) investigated here was shown previously to bind to lipid-linked oligosaccharides 3-O, 4-O and 6-O sulphated at galactose, such as sulphatides and a mixture of 3-sulphated Le(a)/Le(x) type tetrasaccharides isolated from ovarian cystadenoma, as well as to the HNK-1 glycolipid with 3-O sulphated glucuronic acid. In the present study, the L-selectin investigated in both chromatogram binding and plastic microwell binding experiments using neoglycolipids was found to bind to the individual 3-sulphated Le(a) and Le(x) sequences (penta-, tetra- and trisaccharides), and with somewhat lower intensities to their non-fucosylated analogues. Glycosaminoglycan disaccharides of keratan sulphate, heparin and chondroitin sulphate types were also bound by L-selectin in one or both assay systems, leading to the conclusion that clustered glycosaminoglycan oligosaccharides with 6-O sulphation of N-acetylgalactosamine, N-acetylglucosamine or glucosamine, 4-O sulphation of N-acetylgalactosamine, 2-O sulphation of uronic acid, N-sulphation of glucosamine and, to a lesser extent, the non-sulphated uronic acid-containing disaccharides, can support L-selectin adhesion. As inflammatory chemokines (short-range stimulators of lymphocyte migration which trigger integrin activation) are known to bind to endothelial glycosaminoglycans, we propose that the binding of the lymphocyte membrane L-selectin to endothelial glycosaminoglycans may provide a link between the selectin-mediated and integrin-mediated adhesion systems in leukocyte extravasation cascades. The possibility is also raised that lymphocyte L-selectin interactions with glycosaminoglycans may contribute to pathologies of glycosaminoglycan-rich tissues, e.g. cartilage loss in rheumatoid arthritis and inflammatory lesions of the cornea.
TL;DR: It is shown that the specificity of the enzyme in normal rat and human colonic mucosa requires all the substituents of the GalNAc-sugar ring of substrates for maximal activity, and the lack of core 3 beta 3-Glc NAc-T activity in cancer cell lines may be due to cell transformation or cell culturing.
Abstract: UDP-GlcNAc: GalNAc-R beta 3-GlcNAc-transferase (core 3 beta 3-GlcNAc-T, where GlcNAc is N-acetyl-D-glucosamine, GalNAc is N-acetyl-D-galactosamine and T is transferase) is expressed in a tissue-specific fashion and is high in normal colonic tissue, but downregulated in colon cancer. To further study the control of this enzyme, we examined the activity in pig, rat and human colonic tissues, and several human cancer cell lines. The enzyme was difficult to solubilize by detergents and was extremely unstable in the solubilized form. Using synthetic derivatives of the GalNAc-R substrate, we showed that the specificity of the enzyme in normal rat and human colonic mucosa requires all the substituents of the GalNAc-sugar ring of substrates for maximal activity. Core 3 beta 3-GlcNAc-T was significantly influenced by the structure of the aglycon group. None of the inactive substrate derivatives could inhibit the activity. N-Iodoacetamido-galactosamine alpha-benzyl was a weak substrate and significantly inhibited the incorporation of GLcNAc into GalNAc alpha-benzyl by human colonic homogenates. Surprisingly, none of the colonic cancer cell lines or any other cancer and leukaemia cells examined exhibited detectable activity of the enzyme, although a number of other glycosyltransferase activities involved in O-glycan biosynthesis were active. Mixing experiments did not reveal an endogenous inhibitor in HL60 cells or an activator of core 3 beta 3-GlcNAc-T in human colonic mucosa. Thus, the lack of core 3 beta 3-GlcNAc-T in human colonic mucosa. Thus, the lack of core 3 beta 3-GlcNAc-T activity in cancer cell lines may be due to cell transformation or cell culturing.
TL;DR: The results of these analyses establish that rhamnogalacturonase A (RGase A) is an endohydrolase that cleaves the -4)-alpha-D-GalpA-(1-2-alpha-L-Rhap glycosidic linkage, thereby generating oligosaccharides terminating at the non-reducing end with a hex-4-enopyranosyluronic acid residue.
Abstract: Rhamnogalacturonan I (RG-I), a major pectic component of the primary walls of plant cells, is believed to play an important role in determining both the structure and functions of the walls. A more detailed structural description of RG-I is likely to lead to a greater understanding of the biological roles of this polysaccharide. Two enzymes secreted by Aspergillus aculeatus that have been cloned and expressed in a fungal system (Kofod et al., J. Biol. Chem., 269, 29182-29189, 1994) cleave the RG-I backbone in an endo fashion and should assist in the further structural characterization of this polysaccharide. We found that both of the available preparations of the cloned enzymes were contaminated with exoglycanases, reducing their utility in structurally characterizing RG-I. We purified the enzymes to apparent homogeneity by ion-exchange chromatography and then used the purified enzymes to generate backbone oligosaccharide fragments from partially debranched sycamore RG-I. The backbone oligosaccharides, which were separated from larger pieces of partially debranched RG-I by gel-permeation chromatography, have been structurally characterized by 1H-NMR spectroscopy, electrospray MS, GC-MS, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and UV spectroscopy. The results of these analyses establish that rhamnogalacturonase A (RGase A) is an endohydrolase that cleaves the -4)-alpha-D-GalpA-(1-2)-alpha-L-Rhap glycosidic linkage. However, the purported rhamnogalacturonase B (RGase B) is, in fact, an endolyase that cleaves the -2)-alpha-L-Rhap-(1-4)-alpha-D-GalpA glycosidic linkage, thereby generating oligosaccharides terminating at the non-reducing end with a hex-4-enopyranosyluronic acid residue.
TL;DR: The biosynthesis of the Lewis X determinant in three strains of Helicobacter pylori has been investigated and the biosynthetic pathway is identical to that found in humans.
Abstract: The biosynthesis of the Lewis X determinant (Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta-) in three strains of Helicobacter pylori has been investigated. Strains UA 861, UA 802 and UA 1182 contain alpha 1,3 fucosyltransferase and beta 1,4 galactosyltransferase activities that synthesize the Lewis X structure by the transfer of monosaccharides from GDP-fucose and UDP-galactose donors, respectively. The enzyme reaction products that formed were characterized by capillary zone electrophoresis and by 1H-NMR spectroscopy. The biosynthetic pathway is therefore identical to that found in humans. In the three strains, the fucosyltransferase and galactosyltransferase activities differed in various cellular fractions. Km values for their donor and acceptor substrates also differed.
TL;DR: Analysis of human-mouse somatic cell hybrids demonstrates that the sialyltransferase genes are dispersed in the human genome, and coding information for SiaT-4a protein is dispersed into seven discrete exon segments in a manner reminiscent of the SIAT-1 gene.
Abstract: The synthesis of alpha 2,3-linked sialic acid to Gal(beta 1,3)GalNAc is mediated by at least three beta-galactoside alpha 2,3-sialyltransferases (EC 2.4.99.4, SiaT-4) that are encoded by three distinct genes. In contrast, only a single gene encodes the beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1, SiaT-1). This report assesses the relationship and nature of the SiaT-4 genes. Analysis of human-mouse somatic cell hybrids demonstrates that the sialyltransferase genes are dispersed in the human genome. The gene for SiaT-4 resides in chromosome 8, that for SiaT-4b resides in p21-p34 of chromosome 1 and that for SiaT-4c in q23.3-qter of chromosome 11. The gene symbols for these genes have been designated SIAT4A, SIAT4B and SIAT4C, respectively. To assess the structural organization of one of the SiaT-4 genes, a human SiaT-4a cDNA from submaxillary glands was isolated and characterized. Rapid amplification of cDNA 5' ends (5'-RACE) analysis indicates an unusually long 1 kb 5'-untranslated leader. The catalytic domain of the cloned sequence was expressed in transfected cells and was shown to be competent in mediating the specific synthesis of sialic acid alpha 2,3 to Gal(beta 1,3)GalNAc-R. Genomic sequences for SiaT-4a were also isolated and examined. The data demonstrate that coding information for SiaT-4a protein is dispersed into seven discrete exon segments in a manner reminiscent of the SiaT-1 gene. Furthermore, as in the SiaT-1 gene, intervening sequences interrupt both sialylmotif domains, regions that are conserved among all known sialyltransferases.
TL;DR: Homotypic aggregation assays indicated that FRras SNA- were more cohesive with each other and adhesion assays showed that they were more adhesive to fibronectin, and using in vitro invasion assays, FRras cells exhibiting a low alpha-2,6-sialylation on their surface were found to have a low invasive potential compared to their counterpart SNA+.
Abstract: Through cloning experiments with the FRras EJ4 cell line, previously described to exhibit a Sambucus nigra agglutinin (SNA)+ phenotype, three clones with a SNA- phenotype were isolated. All the selected SNA+ and SNA- clones expressed the ras oncoprotein and cloned in soft agar with the same efficiency. We were interested in studying the adhesion and invasion properties of the FRras cells presenting a SNA + or - phenotype. They were first compared in their biochemical properties and we found that FRras SNA- were characterized by a low alpha-2,6-sialylation of their cell surface glycoproteins and a low beta-galactoside alpha-2,6 sialytransferase activity. Using in vitro invasion assays, FRras cells exhibiting a low alpha-2,6-sialylation on their surface were found to have a low invasive potential compared to their counterpart SNA+. FRras SNA-clones exhibit a different morphology from that of FRras SNA+ clones. Moreover, homotypic aggregation assays indicated that FRras SNA- were more cohesive with each other and adhesion assays showed that they were more adhesive to fibronectin.
TL;DR: In this article, the authors present kinetic and structural analyses of oligosaccharide material released during glycosylation in permeabilized Chinese hamster ovary cells incubated with sugar nucleotides.
Abstract: This paper presents kinetic and structural analyses of oligosaccharide material released during glycosylation in permeabilized Chinese hamster ovary cells incubated with sugar nucleotides. Permeabilized cells released 30 times more oligosaccharide material than metabolically labelled cells, normalized to the amount of labelled glycoprotein acceptor, making this an amenable system for study. Fifteen to forty per cent of the oligosaccharide material released by permeabilized cells was oligosaccharide-phosphate, depending on the nature and amount of the oligosaccharide-lipids synthesized. The oligosaccharide-phosphates released were recovered in the cytosol, and were exclusively Man2Glc-NAc2P and Man5GlcNAc2P, released from oligosaccharide-lipids thought to be facing the cytosol. In contrast, the structures found as neutral oligosaccharide material were similar to those attached to newly synthesized glycoproteins, indicating that the oligosaccharides were subjected to the same processing enzymes whether or not they were protein bound. Importantly, the kinetics of the transfer to protein and the release of free neutral oligosaccharide were parallel, suggesting that the same enzyme was responsible for both processes. Structural analyses demonstrated that the same Man5GlcNAc2 structure was transferred to protein and released as free oligosaccharide. Neutral oligosaccharides were found in both the cytosol and the pellet; however, oligosaccharides with one GlcNAc residue at the reducing end (OS-Gn1) were found exclusively in the supernate. The major neutral oligosaccharide produced after 2 h of metabolic labelling was Man5GlcNAc and it was found in the cytosol.
TL;DR: Type I type I CDGS patients produce abnormal N-linked oligosaccharides due to impaired biosynthesis of dolichol-oligosACcharide precursors, suggesting increased glycosylation heterogeneity in CDGS compared with normal alpha-1AT.
Abstract: Carbohydrate-deficient glycoprotein syndrome (CDGS) is a rare metabolic disorder presenting in infancy with severe neurologic involvement and variable multisystemic abnormalities. Diagnosis relies upon the detection of abnormal serum glycoprotein isoforms on isoelectric focusing (IEF) gels. Carbohydrate structural analyses were performed on the N-linked oligosaccharides of serum alpha 1-antitrypsin (alpha-1AT) from two Danish children with classical type I CDGS. Following preparative gel electrophoresis of alpha-1AT isoforms, oligosaccharide charge and monosaccharide composition analyses revealed increased glycosylation heterogeneity in CDGS compared with normal alpha-1AT. CDGS alpha-1AT isoforms bore N-glycans co-migrating with monosialylated standards, while normal alpha-1AT oligosaccharides co-migrated with both mono- and disialylated standards. While the monosaccharide contents of normal alpha-1AT isoforms were relatively uniform, those of CDGS alpha-1AT isoforms varied widely, and many were relatively mannose enriched. The mannose-rich oligosaccharides of CDGS alpha-1AT were not typical oligomannose structures since they were not released by endo-beta-N-acetylglucosaminidase H (endo H) digestion. Metabolic labelling of CDGS fibroblasts with [3H]mannose showed lower than normal intracellular total mannose, free mannose and phosphorylated mannose species, as well as diminished [3H]mannose incorporation into dolichol-linked and protein-linked oligosaccharides. In addition, the glycans liberated from CDGS dolichol-linked oligosaccharides were significantly truncated compared with those from normal fibroblasts. These data suggest that our type I CDGS patients produce abnormal N-linked oligosaccharides due to impaired biosynthesis of dolichol-oligosaccharide precursors.
TL;DR: A strategy to sequence the GAG chains of heparin using 1H-NMR and enzymatic methods to determine the structure of the NDBP-octasaccharides.
Abstract: The versatile biological activities of proteoglycans are mainly mediated by their glycosaminoglycan (GAG) components. Unlike proteins and nucleic acids, no satisfactory method for sequencing GAGs has been developed. This paper describes a strategy to sequence the GAG chains of heparin. Heparin, prepared from animal tissue, and processed by proteinases and endoglucuronidases, is 90% GAG heparin and 10% peptidoglycan heparin (containing small remnants of core protein). Raw porcine mucosal heparin was labelled on the amino termini of these core protein remnants with a hydrophobic, fluorescent tag [N-4-(6-dimethylamino-2-benzofuranyl) phenyl (NDBP)-isothiocyanate]. Enrichment of the NDBP-heparin using phenyl-Sepharose chromatography, followed by treatment with a mixture of heparin lyase I and III, resulted in a single NDBP-linkage region tetrasaccharide, which was characterized as deltaUAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp -(1-->O-Ser-NDBP (deltaUAp is 4-deoxy-alpha-L-threo-hex-4-enopyranosyl uronic acid). Several NDBP-octasaccharides were isolated when NDBP-heparin was treated with only heparin lyase I. The structure of one of these NDBP-octasaccharides, deltaUAp2S(1-->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp(1-->3)-beta-D- Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp-(1-->O-Ser NDBP (S is sulphate, Ac is acetate), was determined by 1H-NMR and enzymatic methods. Enriched NDBP-heparin was treated with lithium hydroxide to release heparin, and the GAG chain was then labelled at xylose with 7-amino-1,3-naphthalene disulphonic acid (AGA). The resulting AGA-Xyl-heparin was sequenced on gradient PAGE using heparin lyase I and heparin lyase III. A predominant sequence in heparin at the protein core attachment site was deduced to be -D-GlcNp2S6S(or 6OH)(1-->4)-alpha-L-IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S (or60H) (1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNp2S6S( or 6OH)(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpAc (1- ->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-++ +GlcAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xyl-AGA.
TL;DR: The O-linked oligosaccharides of the jelly coat surrounding the eggs of Xenopus laevis were analysed by 1H-NMR spectroscopy and showed a high species specificity which could support a biological role during the fertilization processes.
Abstract: The O-linked oligosaccharides of the jelly coat surrounding the eggs of Xenopus laevis were analysed by 1H-NMR spectroscopy. Among the 12 neutral oligosaccharide-alditols which have been characterized, three of them possess the following unusual structures: [sequence: see text] As previously observed for six other amphibian species, the carbohydrate chains of the jelly coat of Xenopus eggs display a high species specificity which could support a biological role during the fertilization processes.
TL;DR: The structures of the oligosaccharides released from four NFA-2 samples by hydrazinolysis were studied by means of lectin-affinity column chromatography, endo- and exo-glycosidase digestion, methylation analysis, hydrazinlysis-nitrous acid deamination and electrospray ionization mass spectrometry.
Abstract: Normal faecal antigen-2 (NFA-2) and non-specific cross-reacting antigen-2 (NCA-2), cross-reacting with anticarcinoembryonic antigen (CEA) antibodies, were found in normal human faeces and meconium, respectively. Because NFA-2, NCA-2 and CEA are considered as the same gene products, NFA-2 and NCA-2 should be normal counterparts of CEA produced by colon epithelial cells of normal adults and fetuses, respectively. Comparison of sugar chain structures of these three antigens is indispensable in order to unravel the structural alteration induced by malignant transformation and development of colon epithelial cells. The sugar chain structures of CEA (Yamashita, K. et al., Cancer Res., 47, 3451-3459, 1987) and NCA-2 (Yamashita, K. et al., J. Biol. Chem., 264, 17873-17881, 1989) were previously reported. In this paper, the structures of the oligosaccharides released from four NFA-2 samples by hydrazinolysis were studied by means of lectin-affinity column chromatography, endo- and exo-glycosidase digestion, methylation analysis, hydrazinolysis-nitrous acid deamination and electrospray ionization mass spectrometry. NFA-2 contains 24-27 mol of N-linked sugar chains/molecule, which is similar to NCA-2 (27 mol) and CEA (24-27 mol). In contrast to CEA, which contains approximately 8% high-mannose-type sugar chains, all sugar chains of NFA-2 are mono- to tetra-antennary complex-type chains having four types of tri-mannosyl cores, with or without bisecting N-acetylglucosamine and fucose residues. The structures of their outer chain moieties comprise Gal beta 1-->3(HSO(3-)-->6)GlcNAc, Neu5Ac alpha 2-->3Gal beta 1-->3GlcNAc, Type 1, repeating chain, Type 2, Type 2H, Type 1H, Lex, Lea and Leb antigenic determinants. Approximately 50% of the outer chain moieties of the oligosaccharides of NFA-2 contain Type 1 chain, in contrast to those of CEAs produced by the liver metastases of colon tumours in which only a trace amount of Type 1 chain was detected.
TL;DR: This is the first description of heptose residues occurring as a constituent of S-layer glycoproteins of gram-positive eubacteria and the following repeating unit structure of the glycan chain is proposed.
Abstract: The characterization of the S-layer glycoprotein of Bacillus thermoaerophilus revealed unexpected novelties. The isolation and purification procedure had to be changed due to complete solubility in aqueous buffers of the constituting S-layer protomers. Upon degradation of the S-layer glycoprotein by pronase and purification of the products by gel filtration, ion-exchange chromatography, chromatofocusing and HPLC, one representative glycopeptide fraction was selected for further characterization. From the combined evidence of composition analysis, chemical degradation, NMR spectroscopy experiments and comparison with synthesized model substance, we propose the following repeating unit structure of the glycan chain: -->4)-alpha-L-Rhap-(1-->3)-beta-D-glycero-D-manno-Hepp-(1--> This is the first description of heptose residues occurring as a constituent of S-layer glycoproteins of gram-positive eubacteria.
TL;DR: Several novel pathways in the synthesis of sulphated mucin-type oligosaccharides are demonstrated, indicating that GlcNAc beta 1-6 branching has to precede sulphation in the O-glycan core 1 processing pathway.
Abstract: A novel sulphotransferase (sulpho-T) activity from rat colonic mucosa was characterized using O-glycan core 1 substrate, Gal beta 1-3GalNAc alpha-benzyl. Derivatives of Gal beta 1-3GalNAc- were used to demonstrate that the 3- and 4-hydroxyl of Gal and the 2-acetamido group of the GalNAc residue of Gal beta 1-3GalNAc alpha-benzyl substrates were important for activity. Sulphated product using Gal beta 1-3GalNAc alpha-benzyl as substrate was analysed by ion spray mass spectrometry, methylation analysis, high-pH anion-exchange chromatography and beta-galactosidase digestion. The results suggested that sulphate was added to the 3-position of the Gal residue. The synthesis of core 2 from core 1 by UDP-GlcNAc: Gal beta 1-3GalNAc beta 6-GlcNAc-transferase was inhibited by sulphation of the Gal residue, indicating that GlcNAc beta 1-6 branching has to precede sulphation in the O-glycan core 1 processing pathway. These data demonstrate several novel pathways in the synthesis of sulphated mucin-type oligosaccharides.
TL;DR: Chicken MBP, bound to mannan, activated the classical complement pathway in human serum and indicate the presence of a MBP-like clearance system outside mammals.
Abstract: A chicken serum lectin was isolated by affinity chromatography on TSK-75 beads derivatized with the monosaccharide N-acetyl-D-mannosamine (ManNAc). Serum was applied to the column in a Ca(2+)-containing buffer and proteins were eluted with EDTA. After recalcification, the eluate was passed through a new ManNAc-derivatized column. Bound proteins were eluted with 50 mM ManNAc. Anti-carbohydrate antibodies present in the eluate were removed by passage through a rabbit anti-chicken immunoglobulin derivatized column, and the lectin was further purified by ion-exchange chromatography and gel-permeation chromatography. The purified chicken lectin shows an overall structure similar to mammalian mannan-binding protein (MBP). SDS-PAGE revealed two polypeptides of M(r) 33 and 34 kDa (reduced) with identical sequence for the first 30 NH2-terminal residues. The NH2-terminal sequence shows 43% identity with the human MBP. Like mammalian MBP, the polypeptides of the chicken lectin are degraded by treatment with collagenase. Residues 26-30 (G-L-P(OH)-G-D) are likely to represent the beginning of the collagenous region. Mobilities on SDS-PAGE of the COOH-terminal collagenase-resistant fragment under reduced and non-reduced conditions indicate the presence of intrachain disulphide bonds, as are also found in mammalian MBP. Gel chromatography showed an intact mol. wt of 750 kDa. Binding of the chicken MBP to mannan was inhibited by monosaccharides in the following order of potency: ManNAc > L-fucose > mannose > N-acetylglucosamine. Other monosaccharides inhibited poorly or not at all. Chicken MBP, bound to mannan, activated the classical complement pathway in human serum. Electron micrographs show structures and dimensions resembling human MBP. Overall, the results show that the purified lectin is the chicken homologue to mammalian MBP and indicate the presence of a MBP-like clearance system outside mammals.
TL;DR: Oral treatment of mice, cutaneously infected with herpes simplex virus type 1 (HSV-1) (strain SC16), with the alpha-glucosidase 1 inhibitor 6-O-butanoyl castanospermine (MDL 28,574) produced a significant delay in lesion development and reduced the amount of virus recovered from the brain.
Abstract: Oral treatment of mice, cutaneously infected with herpes simplex virus type 1 (HSV-1) (strain SC16), with the alpha-glucosidase 1 inhibitor 6-O-butanoyl castanospermine (MDL 28,574) produced a significant delay in lesion development and reduced the amount of virus recovered from the brain Virus load in the brains of mice, whose treatment started 2 days prior to infection, was reduced approximately 100-fold when compared to untreated controls Treatment initiated at the time of infection, while less effective than pre-treatment, nevertheless reduced virus recovery from the brain by 10-fold Consistent with its antiviral activity, orally administered MDL 28,574 was rapidly incorporated by brain tissue and mice fed with compound over extended periods maintained relatively high levels of drug at this site
TL;DR: Observations indicate that amino acid residues 3431-3451 of the laminin G2 domain are involved in HNK-1 carbohydrate-mediated cell adhesion.
Abstract: The N-linked HNK-1 carbohydrate expressed by several recognition molecules mediates the adhesion of early post-natal cerebellar neurons to the G2 domain of the terminal globular domain of the laminin alpha 1 chain (H. Hall et al., submitted). To define this binding site more precisely, G2-derived synthetic peptides were used for binding and competition studies. Peptide 5-G2, comprising the amino acid residues 3431-3451 of G2, inhibited the interaction between the HNK-1-carrying glycolipid and laminin in a concentration-dependent and saturable manner. Peptides which overlap only partially with this sequence interfered less. Peptides comprising other amino acid sequences from G2, and peptides derived from G1 and G3 or a scrambled version of peptide 5-G2, did not show significant effects. Direct binding of peptide 5-G2 to the HNK-1 glycolipid was also demonstrated. Furthermore, peptide 5-G2 interfered in a concentration-dependent and saturable manner with the adhesion of early postnatal cerebellar neurons to laminin. These observations indicate that amino acid residues 3431-3451 of the laminin G2 domain are involved in HNK-1 carbohydrate-mediated cell adhesion.
TL;DR: Findings suggest that the active site of alpha AI consists of W188, R74 and Y190, in analogy to the Trp-Arg-Tyr motif of tendamistat, and that the processing of the polypeptide at Asn77 may be necessary to bring these residues in close proximity.
Abstract: Seeds of the common bean contain three homologous proteins: phytohaemagglutinin E, phytohaemagglutinin L and the lectin-like protein alpha-amylase inhibitor (alpha AI). Whereas the active site of lectins has been studied in great detail, there is no information on the active site of the related protein alpha AI, which exerts its biological activity by making a 1:1 complex with alpha-amylase. alpha-amylase inhibitor is synthesized as a 30 kDa precursor glycoprotein that needs to be processed at Asn77 to form an active molecule. Comparison of the amino acid sequence of the bean alpha AI with that of the bacterial amylase inhibitor, tendamistat, suggested that a region around Trp188 might be involved in the inhibitory site. When a three-dimensional model of the bean alpha AI was constructed based on its homology to the legume lectins, this Trp region was alongside Asn77. To test this site hypothesis, mutants of alpha AI were created by site-directed mutagenesis of the cDNA and expressed in transgenic tobacco. The mutant proteins R74N and WSY188-190GNV, as well as the double mutant, were inactive as inhibitors. These findings suggest that the active site of alpha AI consists of W188, R74 and Y190, in analogy to the Trp-Arg-Tyr motif of tendamistat, and that the processing of the polypeptide at Asn77 may be necessary to bring these residues in close proximity.
TL;DR: S.sanguis OMZ 9 binds to exfoliated HBEC in a sialic acid-sensitive manner and is shown to attach in great numbers to cultured cells, and these cells were shown to not express membrane glycoprotein bearing alpha 2,3-sialylated O-linked carbohydrate chains.
Abstract: Streptococcus sanguis colonizes several human oral surfaces, including both hard and soft tissues. Large salivary mucin-like glycoproteins bearing sialic acid residues are known to bind various S.sanguis strains. However, the molecular basis for the adhesion of S.sanguis to human buccal epithelial cells (HBEC) has not been established. The present study shows that S.sanguis OMZ 9 binds to exfoliated HBEC in a sialic acid-sensitive manner. The desialylation of such cells invariably abolishes adhesion of S.sanguis OMZ 9 to the cell surface. A soluble glycopeptide bearing short sialylated O-linked carbohydrate chains behaves as a potent inhibitor of the attachment of S.sanguis OMZ 9 to exfoliated HBEC. The resialylation of desialylated HBEC with CMP-sialic acid and Gal beta 1,3GalNAc alpha 2,3-sialyltransferase specific for O-glycans restores the receptor function for S.sanguis OMZ 9, whereas a similar cell resialylation with the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase specific for N-glycans is without effect. Finally, the same resialylation reaction carried out with CMP-9-fluoresceinyl-sialic acid as a substrate yields exfoliated HBEC bearing fluorescence on a single 23 kDa protein, when using the alpha 2,3-sialyltransferase as the catalyst. The latter finding demonstrates that this 23 kDa cell surface glycoprotein bears NeuNAc alpha 2-3Gal beta 1-3GalNAc O-linked sugar chains, a carbohydrate sequence which is recognized by S.sanguis OMZ 9 on exfoliated HBEC. In similar experiments carried out with a buccal carcinoma cell line termed SqCC/Y1, S.sanguis OMZ 9 did not attach in great numbers to such cultured cells, and these cells were shown to not express membrane glycoprotein bearing alpha 2,3-sialylated O-linked carbohydrate chains.