CHARACTERIZATION OF THE HUMAN PROLYL 4-HYDROXYLASE TETRAMER AND ITS MULTIFUNCTIONAL PROTEIN DISULFIDE-ISOMERASE SUBUNIT SYNTHESIZED IN A BACULOVIRUS EXPRESSION SYSTEM
CHARACTERIZATION OF THE HUMAN PROLYL 4-HYDROXYLASE TETRAMER AND ITS MULTIFUNCTIONAL PROTEIN DISULFIDE-ISOMERASE SUBUNIT SYNTHESIZED IN A BACULOVIRUS EXPRESSION SYSTEM
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DOI：
10.1073/pnas.89.16.7467
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发表时间：
1992-08-15
影响因子：
11.1
通讯作者：
KIVIRIKKO, KI
中科院分区：
文献类型：
--
作者：
VUORI, K;PIHLAJANIEMI, T;KIVIRIKKO, KI
脯氨酰 4-羟化酶 (EC 1.14.11.2) 是一种 α-2-β-2 四聚体，催化胶原蛋白中 4-羟脯氨酸的翻译后形成。该酶可以很容易地解离成其亚基，但所有在体外将解离的亚基与四聚体结合的尝试均未成功。由于相互排斥的选择性剪接，催化重要的 α 亚基的分子克隆已经鉴定出两种类型的 cDNA 克隆。 β 亚基是一种非常不寻常的多功能多肽，与酶蛋白二硫键异构酶 (EC 5.3.4.1) 相同。我们在此报告了杆状病毒载体在草地贪夜蛾昆虫细胞中表达脯氨酰 4-羟化酶的 α 和 β 亚基以及完全活性的酶四聚体。当单独表达β亚基时，在细胞匀浆的0.1% Triton X-100提取物中发现产生的多肽，并且是完全活性的蛋白质二硫键异构酶。当单独表达任一形式的α亚基时，用0.1% Triton X-100只能从细胞匀浆中提取痕量的α亚基，并且需要1% SDS才能获得有效的溶解。这些α亚基没有脯氨酰4-羟化酶活性。当细胞同时感染产生α和β亚基的病毒时，会形成酶四聚体，但大量的α和β亚基仍然不相关。就共底物和肽底物的比活性和动力学常数而言，重组四聚体与从脊椎动物组织中分离的四聚体没有区别。 α-亚基的两种选择性剪接形式产生具有相同催化特性的酶四聚体。杆状病毒表达似乎是大规模生产酶四聚体和详细研究单体缔合机制的绝佳系统。
Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha-2-beta-2 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important alpha-subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The beta-subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4. 1). We report here on expression of the alpha and beta-subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodoptera frugiperda insect cells by baculovirus vectors. When the beta-subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the alpha-subunit was expressed alone, only traces of the alpha-subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These alpha-subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both alpha- and beta-subunit-producing viruses, an enzyme tetramer was formed, but significant amounts of alpha and beta-subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the alpha-subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.