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

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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 O-glycan chains were isolated from JGP by reductive beta-elimination and their structures were determined as: Gal beta 1-->3[NeuAc alpha 2-->6]GalNAcol (= N-acetylgalactosaminitol), Fuc alpha 1-->2Gal beta 1-->3 [NeuAc alpha 2-->6]GalNAcol, Fuc alpha 1-->2Gal beta 1-->3[GlcNAc beta 1-->6]GalNAcol, and Fuc alpha 1-->2Gal beta 1-->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 Ca(2+)-binding property of JGP was studied by equilibrium dialysis. The high Ca(2+)-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-Ca2+) values was observed. This property of JGP is suited to retaining Ca2+ and keeping its concentration at that just necessary for fertilizing sperm.

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