PROTEASES INVOLVED IN REGULATION OF EGG-ENVELOPES DURING FERTILIZATION AND DEVELOPMENT

受精和发育过程中参与卵包膜调节的蛋白酶

• 批准号: 07458193
• 负责人: KATAGIRI Chiaki
• 金额: $ 4.93万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458193/
• 关键词: VITELLINE ENVELOPE; SPERM-VE BINDING; OVIDUCAL PARS RECTA; TRYPSIN-LIKE PROTEASE; HATCHING ENZYME; METALLOPROTEASE; ASTACIN FAMILY; CUB-DOMAIN; タンパク分解活性; 卵膜溶解活性; 抗UVS.2抗体; oviductin; 卵膜ライシン; 卵外被; 輪卵管直部; プロテアーゼ; 分泌顆粒; 遺伝子発現

Functions and molecular entities were studied of proteases that participate in fertilization and embryonic hatching of anuran amphibians. (a) The eggs of Bufo japonicus become fertilizable during passage through the pars recta (PR) portion of oviduct, accompanied by a significant increase of sperm-binding to vitelline envelope (VE). The sperm binding to VE was found to be mediated by carbohydrate moieties of VE,which was exposed as a result of partial hydrolysis of VE by a trypsin-like protease secreted from PR epithelial cells. The method was developed to isolated this protease, and partial amino acid sequences were determined for its further molecular cloning. (b) Using a previously cloned cDNA,UVS.2, as a probe, a 1.8kb insert (XHE) was cloned from a cDNA library from the NF st.25 Xenopus laevis embryos. XHE was found to code for hatching enzyme, since it encoded 514 amino acids including both signal and propeptide sequences, predicted mature enzyme of 425 amino acids containing metalloprotease of astacin family and CUB-domains. The antibodies against bacterially expressed XHE inhibited the VE digesting activity of the hatching medium, and localized in immunocytochemical stainings the reacting molecules specifically on secretory granules of hatching gland cells. Characterization of the hatching enzyme using these antibodies as probe indicated that the 60kDa enzyme consists of 40kDa molecules of protease domain and other portion which may be involved in recognition and/or processing of the substrate VE.

对参与无尾两栖动物受精和胚胎孵化的蛋白水解酶的功能和分子实体进行了研究。(A)东亚钳蝎卵在通过输卵管的直部(PR)部分时变得可受精，同时精子与卵黄膜(VE)的结合显著增加。精子与VE的结合是由VE的碳水化合物部分介导的，这是由于PR上皮细胞分泌的类胰蛋白酶对VE进行部分水解而暴露出来的。建立了分离该酶的方法，并测定了部分氨基酸序列，为进一步的分子克隆奠定了基础。(2)以已克隆的UVS.2为探针，从非洲爪哇胚胎的cDNA文库中克隆了一个1.8kb的插入片段(XHE)。XHE编码孵化酶，编码514个氨基酸，包括信号和前肽序列，预测425个氨基酸组成的成熟酶，含有ASTIN家族的金属蛋白酶和Cub结构域。针对细菌表达的XHE的抗体抑制了孵化液对VE的消化活性，并定位于免疫细胞化学染色中，反应分子特异性地位于孵化腺细胞的分泌颗粒上。以这些抗体为探针的孵化酶鉴定表明，该60 kDa酶由40 kDa的蛋白酶域分子和可能参与底物VE识别和/或处理的其他部分组成。

项目成果

Teshima, K. et al.: "Relative amounts of basic nuclear proteins SP4 and SP5 in Xenopus laevis sperm correlate with gene copy number." Develop. Growth, Differ.38. 161-166 (1996)

Teshima, K. 等人：“非洲爪蟾精子中碱性核蛋白 SP4 和 SP5 的相对含量与基因拷贝数相关。”

Omata, S. & C. Katagiri: "Involvement of carbohydrate moieties of the toad egg vitelline coat in binding with fertilizing sperm." Develop. Growth, Differ.38. 663-672 (1996)

大俣，S.

Fan, T.J.and Ch.Katagiri: "The mode of action on the vitelline envelope of Xenopus hatching enzyme as studied by its two molecular forms" Zool.Sci. 14. 101-104 (1997)

Fan, T.J. 和 Ch.Katagiri：“通过两种分子形式研究非洲爪蟾孵化酶卵黄包膜的作用模式”Zool.Sci。

Tonegawa, A. et al.: "Heart formative factor (s) is localized in the anterior endoderm of early Xenopus neurula." Ruox's Arch. Dev. Biol.205. 282-289 (1996)

Tonekawa, A. 等人：“心脏形成因子位于早期非洲爪蟾神经胚的前内胚层中。”

Katagiri, Ch.et al: "Molecular cloning of Xenopus hatching enzyme and its specific expression in hatching gland cells." Int.J.Dev.Biol. 41, (in press). (1997)

Katagiri, Ch.等人：“非洲爪蟾孵化酶的分子克隆及其在孵化腺细胞中的特异性表达。”