Isolation, structural determination, and calcium‐binding properties of the major glycoprotein present in Bufo japonicus japonicus egg jelly

Abstract

Although the previous studies showed that the jelly coat is essential in anuran fertilization under natural conditions, identification and structural studies of the macromolecules that play functional roles have remained to be elucidated. In the present study we isolated acidic glycoproteins (JGP) from the solubilized egg jelly of Bufo japonicus japonicus, and showed that they were the major non-dialyzable macromolecular components of the jelly coat. JGP was a typical mucin-type glycoprotein, and it showed high degree of polydispersity in molecular masses ranging over 100-4000 kDa, but both amino acid and carbohydrate compositions were practically identical among fractions, suggesting that JGP was composed of a repeating glycoprotein unit. Four types of short 0-glycan chains were isolated from JGP by reductive p-elimination and their structures were determined as:[NeuAca2+6]GalNAcol, Fucal+2Gal~1-3[GlcNAc~l-+6]GalNAcol, and Fucal+2Galpl+3-GalNAcol. These carbohydrate units (about 80% of the mass of JGP) were linked to nearly all the serine and threonine residues which accounted for 55% of total amino acid residues. The Ca2+-binding property of JGP was studied by equilibrium dialysis. The high Ca2+-binding capacity of JGP was abolished by its desialylation of JGP and was highly dependent on the JGP concentration. When the low JGP concentrations as in the hydrated Bufo jelly were used, a 50% increment of both n (the number of binding sites) and Kd (the dissociation constant of JGP-Ca") values was observed. This property of JGP is suited to retaining Ca2+ and keeping its concentration at that just necessary for fertilizing sperm.

Structure of Six 3‐Deoxy‐d‐Glycero‐d‐Galacto‐Nonulosonic Acid(Kdn)‐Containing Oligosaccharide‐Alditols Released from Oviduct Secretions of Ambystoma Maculatum

“…More generally, it has been demonstrated that the polyanionic mucins surrounding amphibian eggs retain Ca2+ and maintain the Caz+ concentration necessary for successful fertilization [5]. The present study describes new carbohydrate sequences characteristic of the Urodeles Ambystoma maculatum.…”

“…We have shown [2-41 that the carbohydrate structures are highly species specific and exhibit new and unusual features. poraria), whereas NeuAc is present in egg jelly coat mucins of Bufo japonicus [5] and Bufo bufo (Plancke, Y., Wieruszeski, J.-M. and Strecker, G., unpublished results).…”

Structure of three Kdn‐containing oligosaccharide‐alditols released from oviducal secretions of Ambystoma tigrinum: characterization of the carbohydrate sequence Fuc (α1–5) [Fuc (α1–4)] Kdn (α2—3/6)

“…Exceptions are represented by Bufo japonicus [6] and Bufo bufo (personal results), the mucins of which contain exclusively NeuAc. Another characteristic is the presence of Kdn substituted with Fuc, which implies the existence of amphibian-specific fucosyltransferases.…”

“…Studies have shown that they are implicated to play important role in fertilization, involving capacitation of the sperm, induction of the acrosome reaction, block of polyspermy and recognition of homologous species [1][2][3]. Recent works have demonstrated that the carbohydrate chains of these mucins are highly species-specific [4][5][6], that should support the observed species-specificity of gamete interaction. The structural analysis of these components can also furnish the basis of a molecular taxonomy.…”

Profiling the morphological distribution of O-linked oligosaccharides

“…It has been established that in amphibians the fertilization process is dependent upon the presence of jelly coat materials [1]. The proposed biological roles of egg jelly coat may include protection of eggs against pathogens, induction of sperm-egg binding/recognition, initiation of sperm capacitation and/or acrosome reaction, prevention of species cross-fertilization, and blockage of polyspermy [2][3][4][5][6][7][8][9][10][11]. These functions appear to be associated with particular jelly coat layers and are not properties of the jelly coat as a whole.…”