scholarly journals Structural analysis of a new series of oligosaccharide-alditols released by reductive β-elimination from oviducal mucins of Rana utricularia

1998 ◽  
Vol 330 (1) ◽  
pp. 469-478 ◽  
Author(s):  
Willy MORELLE ◽  
Gérard STRECKER
Keyword(s):  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amphibian Species ◽  
Structural Investigations ◽  
Jelly Coat ◽  
Specific Material ◽  
Species Specific ◽  
Carbohydrate Chains ◽  
Rana Utricularia

Egg jelly coats from Rana utricularia are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they pass along the different oviducal portions. In this study, carbohydrate chains of the jelly coat surrounding the eggs of R. utricularia were released by alkali/borohydride treatment. Fractionation of O-linked oligosaccharide-alditols was achieved by a combination of chromatographic techniques comprising anion-exchange chromatography, gel-permeation chromatography and HPLC on a silica column bonded with aminopropyl groups. Structural characterization was performed by one- and two-dimensional 1H-NMR spectroscopy in combination with matrix-assisted laser-desorption ionization-time of flight MS and methylation analysis. Ten oligosaccharide structures possessing a core consisting of Galβ(1 → 3)GalNAc-ol with or without branching through a GlcNAc residue linked β(1 → 6) to the GalNAc residue (core type 2 or core type 1 respectively) are described. The most representative carbohydrate sequences are: GlcNAc(β1-3)[Fuc(α1-4)]GlcNAc, GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc(β1-3)GlcNAc and Gal(β1-3)GlcNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc. The carbohydrate chains isolated from R. utricularia are quite different from those found in other amphibian species, in which the presence of species-specific material has been characterized. Since the jellies surrounding amphibian eggs are involved in egg-sperm interactions, these structural investigations can provide biochemical support for investigation of the fertilization process.

scholarly journals Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue

1990 ◽  
Vol 269 (1) ◽  
pp. 55-59 ◽  
Author(s):  
J M Dickenson ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Ester Group ◽  
Intervertebral Discs ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Linkage Region ◽  
Gel Permeation ◽  
Electron Micro Probe Analysis

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].

scholarly journals Osmoregulated Periplasmic Glucans ofErwinia chrysanthemi

2001 ◽  
Vol 183 (10) ◽  
pp. 3127-3133 ◽  
Author(s):  
Virginie Cogez ◽  
Philippe Talaga ◽  
Jerome Lemoine ◽  
Jean-Pierre Bohin
Keyword(s):  
High Performance ◽  
Large Fraction ◽  
Compositional Analysis ◽  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Variable Number ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Trichloracetic Acid ◽  
Laser Desorption Mass Spectrometry

ABSTRACT We report the initial characterization of the osmoregulated periplasmic glucans (OPGs) of Erwinia chrysanthemi. OPGs are intrinsic components of the bacterial envelope necessary to the pathogenicity of this phytopathogenic enterobacterium (F. Page, S. Altabe, N. Hugouvieux-Cotte-Pattat, J.-M. Lacroix, J. Robert-Baudouy and J.-P. Bohin, J. Bacteriol. 183:0000–0000, 2001 [companion in this issue]). OPGs were isolated by trichloracetic acid treatment and gel permeation chromatography. The synthesis of these compounds appeared to be osmoregulated, since lower amounts of OPGs were produced when bacteria were grown in media of higher osmolarities. However, a large fraction of these OPGs were recovered in the culture medium. Then, these compounds were characterized by compositional analysis, high-performance anion-exchange chromatography, matrix-assisted laser desorption mass spectrometry, and 1H and 13C nuclear magnetic resonance analyses. OPGs produced by E. chrysanthemi are very heterogeneous at the level of both backbone structure and substitution of these structures. The degree of polymerization of the glucose units ranges from 5 to 12. The structures are branched, with a linear backbone consisting of β-1,2-linked glucose units to which a variable number of branches, composed of one glucose residue, are attached by β-1,6 linkages in a random way. This glucan backbone may be substituted by O-acetyl andO-succinyl ester-linked residues.

scholarly journals Xyloglucan endotransglycosylase: evidence for the existence of a relatively stable glycosyl–enzyme intermediate

1998 ◽  
Vol 330 (3) ◽  
pp. 1475-1480 ◽  
Author(s):  
Zdena SULOVÁ ◽  
Miriam TAKÁČOVÁ ◽  
M. Nancy STEELE ◽  
C. Stephen FRY ◽  
Vladimír FARKAŠ
Keyword(s):  
Complex Formation ◽  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glycosidic Bond ◽  
Xyloglucan Endotransglycosylase ◽  
Anomeric Configuration ◽  
Gel Permeation ◽  
Covalently Linked ◽  
Enzyme Intermediate

Xyloglucan endotransglycosylases (XETs) catalyse the breakdown of xyloglucan molecules predominantly by transglycosylation. In this process, fragments of cleaved polysaccharide are preferentially transferred to other xyloglucan molecules or their oligosaccharide subunits, with overall retention of the anomeric configuration of the glycosidic bond. In accordance with the theory, we propose that the cleavage and re-formation of the glycosidic bond in xyloglucan involves the formation of a glycosyl-enzyme intermediate which decomposes by transfer of the glycosyl moiety to a suitable carbohydrate acceptor. XETs from nasturtium seed cotyledons, mung bean hypocotyls and cauliflower florets interacted with xyloglucan to form complexes of high Mr as judged by gel-permeation chromatography. The nasturtium enzyme also showed evidence of XET-xyloglucan complex-formation according to anion-exchange chromatography and adsorption of the complex to filter paper on the basis of affinity of its xyloglucan moiety for cellulose. The XET-xyloglucan complex was stable in water, 6 M urea and acidic and alkaline buffers (pH 2.5-9.5), but readily decomposed by transferring its glycosyl moiety to xyloglucan-derived oligosaccharides or by incubation with the strong nucleophile imidazole at pH 3.8-9.6. These results strongly support the assumption that XET forms a relatively stable covalently linked glycosyl-enzyme intermediate.

scholarly journals Purification and characterization of an extracellular fragment of the sea urchin egg receptor for sperm.

1990 ◽  
Vol 111 (6) ◽  
pp. 2951-2959 ◽  
Author(s):  
K R Foltz ◽  
W J Lennarz
Keyword(s):  
Sea Urchin ◽  
Specific Binding ◽  
Specific Interaction ◽  
Gel Filtration ◽  
Extracellular Domain ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Species Specificity ◽  
Enzyme Treatment ◽  
Species Specific

Fertilization in the sea urchin involves species-specific interaction between the ligand bindin on the surface of acrosome-reacted sperm and a receptor of high molecular weight on the surface of the egg. Efforts to understand this interaction and the resultant signal transduction events leading to egg activation have been limited because of the large size and extreme insolubility of the intact receptor on the egg surface. Earlier work suggested that an alternative strategy would be to isolate proteolytic fragments of the extracellular domain of this receptor. Consequently, we have treated S. purpuratus eggs with a specific protease, lysylendoproteinase C. This enzyme treatment abolished the ability of eggs to bind sperm and resulted in the release of proteolytic fragments that bound to sperm and showed inhibitory activity in a fertilization bioassay. One of these fragments, presumed to be a fragment of the extracellular domain of the receptor, was purified to homogeneity by gel filtration and anion exchange chromatography and shown to be a 70-kD glycosylated protein. Several lines of evidence support the contention that this fragment is derived from the receptor. First, the fragment inhibited fertilization species specifically. Second, species specific binding of the 70-kD glycoprotein to acrosome-reacted sperm was directly demonstrated by using 125I-labeled receptor fragment. Third, the fragment exhibited the same species specificity in binding to isolated bindin particles. Species specificity was abolished by Pronase digestion of the fragment. This observation supports the hypothesis that although binding is mediated by the carbohydrate moieties, species specificity is dependent on the polypeptide backbone. The availability of a structurally defined fragment of the receptor will facilitate further studies of the molecular basis of gamete interaction.

Species specificity of O-linked carbohydrate chains of the oviducal mucins in amphibians: structural analysis of neutral oligosaccharide alditols released by reductive β-elimination from the egg-jelly coats of Rana clamitans

2002 ◽  
Vol 363 (3) ◽  
pp. 457-471 ◽  
Author(s):  
Florence DELPLACE ◽  
Emmanuel MAES ◽  
Jérôme LEMOINE ◽  
Gérard STRECKER
Keyword(s):  
Rana Clamitans ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Jelly Coat ◽  
Neutral Compounds ◽  
Reductive Treatment ◽  
Species Specific ◽  
Coat Material ◽  
Neutral Oligosaccharide ◽  
Carbohydrate Chains

The extracellular matrix (the so-called jelly coat) surrounding amphibian eggs mainly comprises highly O-glycosylated proteins. These oviducal mucins have an important role in the fertilization process, and their carbohydrate chains are remarkably species-specific. Alkaline reductive treatment of the jelly-coat material of the frog Rana clamitans led to the release of oligosaccharide alditols. The neutral oligosaccharide alditols were fractionated and purified by successive chromatographic techniques. The structures of 27 of them, ranging from three to sixteen monosaccharides, were established by a combination of NMR spectroscopy, methylation analyses and matrix-assisted laser-desorption ionization—time of flight MS. Typically, some of the neutral compounds appeared to possess the core structure: Gal(β1–3)[GlcNAc(β1–6)]Gal(β1–3)[GlcNAc(β1–6)]GalNAc-ol (where GalNAc-ol represents N-acetylgalactosaminitol). Moreover, a novel type of chain termination, characterized by an unusual sequence {Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–3/4)} was observed. Indeed, the most complex representative structure of this series was found to be: Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–3)[Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–3)[Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–4)GlcNAc(β1–6)]GalNAc-ol.

Characterization of a spore protein inducing factor from Dictyostelium discoideum

1988 ◽  
Vol 89 (3) ◽  
pp. 387-395
Author(s):  
F. P. GIBSON ◽  
B. DAVID HAMES
Keyword(s):  
Protein Synthesis ◽  
Coat Protein ◽  
Anion Exchange ◽  
Dictyostelium Discoideum ◽  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Acid Group ◽  
Exchange Chromatography ◽  
A Minor

Spore coat protein synthesis during development by submerged pseudoplasmodia of Dictyostelium discoideum requires a low molecular weight factor secreted by cells incubated at high density inbuffer. The further characterization of this sporeprotein inducing factor (SPIF) is reported. Its behaviour during anion-exchange chromatography and the loss of activity upon esterification suggests the presence of a carboxylic acid group essential for biological activity. Gel permeation chromatography resolves a major SPEF activity with Mr ∼ 160–200 and a minor activity with Mr ∼ 340–420. Anion-exchange HPLC further resolves the major SPIF activity into four components, one major and three minor. Methionine, analogues of methionine, and precursors of methioninebio synthesis are all effective in maintainingspore coat protein synthesis. Condition edmedium contains methionine at a concentration sufficient to account for its SPIF activity and this activity is abolished by cyanogen bromide treatment. These results indicate that SPIF is eithermethionine or a close analogue of methionine.

scholarly journals Structural analysis of the oligosaccharide-alditols released by reductive β-elimination from the jelly coat of Rana utricularia eggs

1997 ◽  
Vol 321 (3) ◽  
pp. 879-887 ◽  
Author(s):  
Willy MORELLE ◽  
Gérard STRECKER
Keyword(s):  
Nmr Spectroscopy ◽  
Structural Analysis ◽  
Blood Group ◽  
1H Nmr ◽  
1H Nmr Spectroscopy ◽  
Blood Group A ◽  
Jelly Coat ◽  
Group A ◽  
Carbohydrate Chains ◽  
Rana Utricularia

The O-linked oligosaccharides of the jelly coat surrounding the eggs of Rana utricularia were analysed by 1H-NMR spectroscopy. Comparison of their structures with those characterized from seven other amphibians confirms that the carbohydrate chains of the jelly coat mucins are markers of the species. The new sequence GlcNAc(β1-3)GlcNAc(β1-6)[Gal(β1-3)]GalNAc-ol is characteristic of Rana utricularia. The presence of blood group A determinants constitutes the main feature of this mucin.

Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia

2020 ◽  
Author(s):  
Ian Sims ◽  
A Bacic
Keyword(s):  
Uronic Acid ◽  
Cell Suspension Cultures ◽  
Anion Exchange Chromatography ◽  
Nicotiana Plumbaginifolia ◽  
Suspension Cultures ◽  
Exchange Chromatography ◽  
Arabinogalactan Protein ◽  
Extracellular Polysaccharides ◽  
Selective Precipitation ◽  
The Individual

The soluble polymers secreted by cell-suspension cultures of Nicotiana plumbaginifolia contained 78% carbohydrate, 6% protein and 4% inorganic material. The extracellular polysaccharides were separated into three fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7 and the individual polysaccharides in each fraction were then isolated by selective precipitation and enzymic treatment. Monosaccharide and linkage compositions were determined for each polysaccharide after reduction of uronic acid residues and the degree of esterification of the various uronic acid residues in each polysaccharide was determined concurrently with the linkage types. Six components were identified: an arabinoxyloglucan (comprising 34% of the total polysaccharide) and a galactoglucomannan (15%) in the unbound neutral fraction, a type II arabinogalactan (an arabinogalactan-protein, 11%) and an acidic xylan (3%) in the first bound fraction, and an arabinoglucuronomannan (11%) and a galacturonan (26%) in the second bound fraction. © 1995.

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