Oviducal Mucins Research Articles

Identification of the blood group Lewis a determinant in the oviducal mucins of Xenopus tropicalis

The amphibian Xenopus tropicalis appears an increasingly appealing model for both genetic and developmental biology studies, compared to the related species Xenopus laevis. Study of the glycosylation pattern of its secreted glycoproteins revealed that this species synthesizes large amounts of Lewis a epitope, whereas this motif has previously only been identified in animals within the primate lineage. The use of 1H-nuclear magnetic resonance spectroscopy enabled us to resolve the sequence of three Lewis a-bearing O-linked glycans associated with oviducal secretions, out of which one contained the novel sequence Gal(β1–3)GlcNAc(β1–6)GalNAc-ol. These structural data suggested the emergence of an α1,4-fucosyltransferase activity in animals outside the primate lineage. On this basis, the screening of a X. tropicalis GenBank database with human Lewis-fucosyltransferase sequences revealed the occurrence of a putative fucosyltransferase gene that presented an unusual acceptor motif.

Comparative study of carbohydrate chains released from the oviducal mucins of the two very closely related amphibian species Bombina bombina and Bombina variegata

The eggs of amphibians are surrounded by an extracellular matrix, termed jelly coat, which is mainly composed of hydrated mucin-type glycoproteins. These highly glycosylated molecules are synthesized by the oviduct and play an important role in the fertilization process. From a structural and chemical point of view, these oviducal mucins are very different from one species to another and they could be involved in the species-specificity of gamete interactions or could influence the parasite tropism. Bombina bombina and Bombina variegata are the two most closely related species within the genus, which hybridize readily in nature. Divergence occurred during geographic isolation estimated at 2-7 million years ago. The oviducal mucins of these species have been studied at the carbohydrate level, and the primary structures of 28 compounds have been established by NMR spectroscopy. The carbohydrate chains released from the oviducal mucins of the two species were similar and characterized by the common sequences GlcNAc(β1-3)[Fuc(α1-4)]GlcNAc(β1-6) and GlcNAc(α1-4)Gal(β1-4)Gal(β1-3) attached to GalNAc-ol (core 2). Nevertheless, some differences confirmed the strict species-specificity of amphibian oviducal carbohydrate chains observed previously. On the one hand, the presence of βGal 1,4-linked to βGlcNAc in B. bombina, but not in B. variegata, can indicate that β4GalT: βGlcNAc and β4GalT: βGal are two distinct glycosyltransferases. On the other hand, deaminoneuraminic acid (Kdn) is present in B. bombina, and N -glycolylneuraminic acid (NeuGc) in B. variegata. Although the enzymes involved in the biosynthesis of Kdn are not as well characterized, it can be suggested that at least one step of the biosynthetic pathway of NeuAc has been disrupted, leading the B. bombina oviducal NeuAc-9-synthase to use Man-6-P as a substrate, instead of ManNAc-6-P.

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

A combination of ion-exchange chromatography and high performance liquid chromatography (HPLC) has been used to separate the reduced oligosaccharides produced by alkaline borohydride degradation of oviducal mucins obtained from the jelly coat of Rana dalmatina. The primary structures of 26 O-glycans were determined by one-dimensional and two-dimensional 1H and 1H 13C NMR spectroscopy. As observed for 20 other amphibian species, these carbohydrate chains are highly species-specific. The main typical feature of the species R. dalmatina consists in the presence of the backbone Gal(β1-3)[Gal(β1-4)]Gal(β1-3)GalNAc-ol, previously observed among Ranidae, such as R. temporaria and R. ridibunda. Nevertheless, the nature of carbohydrates present at the periphery of the glycans perfectly differentiates the three species.

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

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(beta1-3)[GlcNAc(beta1-6)]Gal(beta1-3)[GlcNAc(beta1-6)]GalNAc-ol (where GalNAc-ol represents N-acetylgalactosaminitol). Moreover, a novel type of chain termination, characterized by an unusual sequence [Fuc(alpha1-2)Gal(alpha1-3)Gal(alpha1-4)Gal(beta1-3/4)] was observed. Indeed, the most complex representative structure of this series was found to be: Fuc(alpha1-2)Gal(alpha1-3)Gal(alpha1-4)Gal(beta1-3)[Fuc(alpha1-2)Gal(alpha1-3)Gal(alpha1-4)Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-3)[Fuc(alpha1-2)Gal(alpha1-3)Gal(alpha1-4)Gal(beta1-4)GlcNAc(beta1-6)]GalNAc-ol.

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

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.

Species-specificity of amphibia carbohydrate chains: the Bufo viridis case study

The jelly coat surrounding the eggs of amphibia is composed of oviducal mucins and plays an important role in the fertilization process. From a structural and chemical point of view, these jellies are very different from one species to another. Bufo viridis is the 13th amphibia species studied in term of carbohydrate structural analysis. The oligosaccharides have been released from the oviducal mucins by reductive β elimination, purified by various chromatography procedures and analyzed by 1H and 13C 1D–2D NMR spectroscopy. Among the 15 compounds, ten have novel structures, although they possess some well-known structural patterns as blood group epitopes (Le x, Le y) or other sequences already observed in other amphibia species. These results reinforce our hypothesis about the strict species-specificity of these carbohydrate chains. It must be noted that such species-specificity does not depend on one particular monosaccharide but it is rather due to a set of particular tri- or tetrasaccharide sequences. Hence, B. viridis species could be characterized by the simultaneous presence of a 2,3,6-trisubstituted galactosyl residue, the GlcNAc(β1–3)[Fuc(α1–4)]GlcNAcβ sequence and the Le x, Le y or Cad determinants. The anionic charge of the oligosaccharides is carried only by sialic acid α-(2→6)-linked to GalNAc-ol residue as in Bufo bufo or in Bufo arenarum.

Acquisition of species-specific O-linked carbohydrate chains from oviducal mucins in Rana arvalis. A case study.

The extracellular matrix surrounding amphibian eggs is composed of mucin-type glycoproteins, highly O-glycosylated and plays an important role in the fertilization process. Oligosaccharide-alditols were released from the oviducal mucins of the anuran Rana arvalis by alkali-borohydride treatment in reduced conditions. Neutral and acidic oligosaccharides were fractionated by ion-exchange chromatographies and purified by HPLC. Each compound was identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometry, NMR spectroscopy, electrospray ionization-tandem mass spectroscopy (ESI-MS/MS) and permethylation analyses. This paper reports on the structures of 19 oligosaccharide-alditols, 12 of which have novel structures. These structures range in size from disaccharide to octasaccharide. Some of them are acidic, containing either a glucuronic acid or, more frequently, a sulfate group, located either at the 6 position of GlcNAc or the 3 or 4 positions of Gal. This latter sulfation is novel and has only been characterized in the species R. arvalis. This structural analysis led to the establishment of several novel carbohydrate structures, demonstrating the structural diversity and species-specificity of amphibian glycoconjugates.

Structural analysis of 20 oligosaccharide-alditols released from the jelly coat of Rana palustris eggs by reductive beta-elimination characterization of the polymerized sequence [Gal(beta1, 3)GalNAc(alpha1-4)]n.

The eggs of amphibians are surrounded by three to eight layers of jelly coats. This extracellular matrix is mainly composed of hydrated mucin-type glycoproteins. These highly glycosylated molecules are synthesized by oviduct and play an important role in the fertilization process. Recent structural analyses have shown the strict species-specificity of the O-linked oligosaccharides which constitute 60-70% of these oviducal mucins. Consequently, these carbohydrate chains represent new phenotypic markers, and from a biological point of view, can influence parasite tropism or can be involved in species-specific interaction of gametes. The primary structure of 20 oligosaccharide-alditols, released by alkali/borohydride treatment from the mucin of Rana palustris egg jelly coats, was established by 1H and 13C-NMR analysis. Thirteen of these components possess new structures and the polymerization of the sequence Gal(beta1-3)GalNAc(alpha1-4) characterizes the species-specificity of R. palustris.

Structural analysis of a new series of oligosaccharide-alditols released by reductive beta-elimination from oviducal mucins of 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 Galbeta(1-->3)GalNAc-ol with or without branching through a GlcNAc residue linked beta(1-->6) to the GalNAc residue (core type 2 or core type 1 respectively) are described. The most representative carbohydrate sequences are: GlcNAc(beta1-3)[Fuc(alpha1-4)]GlcNAc, GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)GlcNAc(beta1-3)GlcNAc and Gal(beta1-3)GlcNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)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.

Isolation of the O-glycosidically linked oligosaccharides obtained by alkaline borohydride degradation from oviducal mucins of the toad Bufobufo

A combination of normal-phase high-performance liquid chromatography (HPLC) on amino-bonded silica and reversed-phase HPLC on octadecylsilica has been used to separate the reduced oligosaccharides produced by alkaline borohydride degradation of oviducal mucins obtained from the jelly coat of Bufo bufo. The former technique provides suitable separation on the basis of molecular size, while the latter method offers selectivity for stereoisomers. Thirty-four compounds, ranging in size from a trisaccharide to a dodecaoligosaccharide, have been isolated preparatively using a Supelcosyl LC-NH2 normal-phase column eluted with aqueous acetonitrile and a Zorbax ODS reversed-phase column eluted with water.

Structural analysis of oligosaccharide-alditols released by reductive β-elimination from oviducal mucins of Rana dalmatina

The O-linked oligosaccharides of the jelly coat surrounding the eggs of Rana dalmatina were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by consecutive normal-phase high-performance liquid chromatography on a silica-based alkylamine column. The structures of the oligosaccharide-alditols were determined by 400-MHz 1H-NMR spectroscopy in combination with matrix assisted laser desorption ionization–time of flight analysis. The five structures were identified range in size from trisaccharides to hexasaccharides, possessing a core consisting of Gal( β1–3)GalNAc-ol (core type 1). Novel oligosaccharide-alditols are: The carbohydrate chains isolated from Rana dalmatina are different from those found in other amphibian species, in which the presence of species-specific material has been characterized. Since the role of carbohydrates appears more and more apparent during the fertilization process, the biodiversity of the O-linked oligosaccharides could support such a biological role.

Primary structure of seven sulfated oligosaccharide-alditols released by reductive β-elimination from oviducal mucins of Rana temporaria characterization of the sequence HSO 3(3)GlcA(β1–3)Gal

The mucins isolated from Rana temporaria egg jelly coats were found to be composed of Gal, Fuc, GlcNAc, Ga1NAc and GlcA acid. The primary structure of seven sulfated oligosaccharide-alditols was obtained by 1D/2D NMR analyses ( 1H- 13C). The results show the presence of the sulfated monosaccharides HSO 3(3)Gal, HSO 3(6)GlcNAc and HSO 3(3)GlcA. The sequence HSO 3(3)GlcA(β1–3)Gal, which constitutes the major determinant of the HNK-1 oncofoetal epitope, was characterized.

Structural analysis of oligosaccharide-alditols released by reductive beta-elimination from oviducal mucins of Bufo bufo: characterization of the carbohydrate sequence Gal(alpha1-3)GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal.

Several tri- to hexasaccharide-alditols of the jelly coat surrounding the eggs of Bufo bufo were studied by methylation analysis, MALDI-TOF mass spectrometry, and 1H-NMR spectroscopy. As observed for six other amphibian species, these carbohydrate chains are highly species-specific. The main characteristics of the species Bufo bufo consists in the presence of the new carbohydrate sequence Gal(alpha1-3)GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal, in which a blood group A determinant is substituted with an external alpha1-3 linked galactose unit. Since the role of carbohydrates appears more and more apparent during the fertilization processes, the species-specificity of these carbohydrate moieties should be relevant to the species-specific gamete recognition which characterizes most amphibians.

Structural analysis of the oligosaccharide-alditols released by reductive beta-elimination from oviducal mucins of Rana temporaria.

The carbohydrate chains of the mucins which constitute the jelly coat surrounding the eggs of Rana temporaria were released by alkaline borohydride treatment. Neutral and acidic oligosaccharide-alditols were purified by ion-exchange chromatography and HPLC. From the structural analysis, based upon 1H and 13C-NMR spectroscopy in combination with MALDI-TOF, the following glycan units are proposed.

Structural analysis of hexa to dodecaoligosaccharide-alditols released by reductive beta-elimination from oviducal mucins of Bufo bufo.

Hexa to dodecasaccharide-alditols of the jelly coat surrounding the eggs of the toad Bufo bufo were studied by methylation analysis, MALDI-TOF mass spectrometry, and H-NMR spectroscopy. These highly species-specific carbohydrate chains exhibit new structural features, such as the elongation of the blood group A determinant with an external alpha-1,3-linked galactose unit, or ramification belonging from a fucosylated galactose. The most representative oligosaccharide-alditol of the series was defined as following: [structure in text]. Since the jellies surrounding amphibian eggs are involved in egg-sperm interactions, these structural investigations can provide biochemical support for exploring the fertilization process.