Isolation, structural determination, and calcium-binding properties of the major glycoprotein present in Bufo japonicus japonicus egg jelly.
Isolation, structural determination, and calcium-binding properties of the major glycoprotein present in Bufo japonicus japonicus egg jelly.
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蟾蜍蛋冻中主要糖蛋白的分离、结构测定和钙结合特性。
DOI：
10.1111/j.1432-1033.1994.tb18986.x
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Y. Inoue
中科院分区：
文献类型：
--
作者：
Yasushi Shimoda;K. Kitajima;Sadako Inoue;Y. Inoue
虽然以往的研究表明，果冻外套是必不可少的无尾两栖类受精在自然条件下，鉴定和结构的大分子发挥功能的作用仍有待阐明。从溶解的中华大蟾蜍卵冻中分离出酸性糖蛋白（JGP），并表明它们是冻衣的主要非透析性大分子成分。JGP是一种典型的粘蛋白型糖蛋白，其分子量在100-4000 kDa范围内具有高度的多分散性，但各组分的氨基酸和碳水化合物组成几乎相同，表明JGP是由一个重复的糖蛋白单元组成的。通过还原性β-消除从JGP中分离出四种类型的短O-聚糖链，其结构确定为：半乳糖β 1-->3[NeuAc α 2-->6]半乳糖（= N-乙酰氨基半乳糖醇），Fuc α 1->2Gal β 1->3 [NeuAc α 2->6]GalNAcol，Fuc α 1->2Gal β 1->3[GlcNAc β 1->6]GalNAcol，和Fuc α 1->2Gal β 1->3-GalNAcol。这些碳水化合物单元（约占JGP质量的80%）与几乎所有的丝氨酸和苏氨酸残基连接，占总氨基酸残基的55%。用平衡透析法研究了JGP与Ca（2+）的结合特性。JGP的高钙结合能力被其去唾液酸化作用所消除，并且高度依赖于JGP的浓度。当使用低JGP浓度时，如在水合蟾蜍果冻中，观察到n（结合位点的数目）和Kd（JGP-Ca 2+的解离常数）值增加50%。JGP的这种特性适合于保留Ca 2+并将其浓度保持在精子所需的浓度。
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.