Post-Translational Modification by Tyrosine Sulfation and Its Functions

酪氨酸硫酸化翻译后修饰及其功能

• 批准号: 03660093
• 负责人: SUIKO Masahito
• 金额: $ 1.41万
• 依托单位: Miyazaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03660093/
• 关键词: Tyrosine Sulfation; Posttranslational Modification; Golgi body; Bovine Liver; Sulfotransferase; Hirudin; Thrombin inhibitor; Active Sulfate; リコンビナントタンパク質; タンパク質; オリゴペプチド; 遺伝子工学

Tyrosine sulfation is an important post-translational modification with a range of functional relevancies, and for those proteins that are sulfated in nature, this modification could be indispensable for the implication of their function. It is known that when sulfated proteins are expressed in bacteria or yeast, the resultant proteins are not tyrosine-sulfated due to the lack of this modification in those vectors, and hence the in vitro tyrosine-sulfation of recombinant proteins by tyrosylprotein sulfotransferase (TPST) would be a valuable tool in converting proteins so produced to their natural form. Hirudin, a potent thrombin inhibitor secreted by the medicinal leech, Hirudo medicinalis, requires sulfation of its 63rd tyrosine for exertion of its fullest activity as a thrombin inhibitor. Recombinant hirudin, expressed in bacteria or yeast is not tyrosine sulfated.For the post-translational modification of recombinant hirudin, TPST was prepared from a bovine liver Golgi preparation. TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15, indicating its specificity for the naturally sulfated tyrosine 63.

酪氨酸硫酸化是一种重要的翻译后修饰，具有一系列的功能相关性，对于那些在自然界中被硫酸化的蛋白质，这种修饰对于其功能的含义可能是不可或缺的。众所周知，当硫酸盐化蛋白在细菌或酵母中表达时，由于在这些载体中缺乏这种修饰，所得到的蛋白不是酪氨酸硫化的，因此，通过酪氨酸蛋白硫转移酶（TPST）在体外对重组蛋白进行酪氨酸硫化将是将蛋白质转化为自然形式的有价值的工具。水蛭素是一种有效的凝血酶抑制剂，由药用水蛭水蛭分泌，水蛭需要磺化其63酪氨酸才能发挥其作为凝血酶抑制剂的最大活性。重组水蛭素，表达在细菌或酵母不是酪氨酸硫酸盐。为了对重组水蛭素进行翻译后修饰，我们从牛肝脏高尔基体中制备了TPST。TPST能够抑制在大肠杆菌和c端水蛭素片段54-65中表达的重组水蛭素变体1 (rHV-1)，但不能抑制n端水蛭素片段1-15，表明其对天然硫酸酪氨酸63的特异性。

项目成果

Masahito Suiko: "Detoxication of Phenolic Xenobiotics in Microsomal Membranes: A Novel Phenol Sulfotransferase Activity in Bovine Liver Microsomes." The 6th China-Japan Symposium on Pesticide Science. 151-155 (1992)

Masahito Suiko：“微粒体膜中酚类异生物质的解毒：牛肝微粒体中的新型酚磺基转移酶活性。”

Jian Rong Han: "PostーTranslational Modification and Binding Properties of Apically Secreted 80 KDa Glycoprotein from Madin Derby Canine Kidney Cells:Similarities to the CーTerminal Portion of the Basolaterally Secreted Fibronectin." Archives of Biochemistr

韩建荣 (Jian Rong Han)：“Madin Derby 犬肾细胞顶端分泌的 80 KDa 糖蛋白的翻译后修饰和结合特性：与基底外侧分泌的纤连蛋白档案的 C 末端部分的相似性。”

MingーCheh Liu: "Identification of Complexes between the TyrosineーOーSulfateーBinding Protein and TyrosineーSulfated Proteins in Bovine Liver Membrane Lysate." Biochemical Journal. 275. 259-262 (1991)

Ming-Cheh Liu：“牛肝膜裂解物中酪氨酸-O-硫酸盐结合蛋白和酪氨酸硫酸化蛋白之间复合物的鉴定”，275。259-262（1991）。

Masahito Suiko: "Detoxication of Phenolic Xenobiotics in Microsomal Membranes:A Novel Phenol Sulfotransferase Activity in Bovine Liver Microsomes." The 6th China-Japan Symposium Series. 151-155 (1992)

Masahito Suiko：“微粒体膜中酚类异生物质的解毒：牛肝微粒体中的新型酚磺基转移酶活性。”

MingーCheh Liu: "Possible Involvement of Protein Tyrosins Sulfation in the Polarized Protein Sorting in FilterーGrown MadinーDarby Canine Kidney(MDCK)Cells." Proceeding of the Fourth Annual Meeting and International Symposium of Japanese Association For Anim

Ming-Cheh Liu：“蛋白质酪氨酸硫酸化可能参与过滤培养的 Madin-Darby 犬肾 (MDCK) 细胞的极化蛋白质分选。”日本动物协会第四届年会暨国际研讨会论文集