Soitamo A J, Rabergh C M, Gassmann M, Sistonen L, Nikinmaa M
Turku Centre for Biotechnology, University of Turku, Abo Akademi University, FIN-20521 Turku, Finland.
J Biol Chem. 2001 Jun 8;276(23):19699-705. doi: 10.1074/jbc.M009057200. Epub 2001 Mar 9.
The mammalian hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that controls the induction of several genes involved in glycolysis, erythropoiesis, and angiogenesis when cells are exposed to hypoxic conditions. Until now, the expression and function of HIF-1alpha have not been studied in fish, which experience wide fluctuations of oxygen tensions in their natural environment. Using electrophoretic mobility shift assay, we have ascertained that a hypoxia-inducible factor is present in rainbow trout cells. We have also cloned the full-length cDNA (3605 base pairs) of the HIF-1alpha from rainbow trout with a predicted protein sequence of 766 amino acids that showed a 61% similarity to human and mouse HIF-1alpha. Polyclonal antibodies against the N-terminal part (amino acids 12-363) and the C-terminal part (amino acids 330-730) of rainbow trout HIF-1alpha protein recognized rainbow trout and chinook salmon HIF-1alpha protein in Western blot analysis. Also, the human and mouse HIF-1alpha proteins were recognized by the N-terminal rainbow trout anti-HIF-1alpha antibody but not by the C-terminal HIF-1alpha antibody. The accumulation of HIF-1alpha was studied by incubating rainbow trout and chinook salmon cells at different oxygen concentrations from 20 to 0.2% O(2) for 1 h. The greatest accumulation of HIF-1alpha protein occurred at 5% O(2) (38 torr), a typical oxygen tension of venous blood in normoxic animals. The protein stability experiments in the absence or presence of a proteasome inhibitor, MG-132, demonstrated that the inhibitor is able to stabilize the protein, which normally is degraded via the proteasome pathway both in normoxia and hypoxia. Notably, the hypoxia response element of oxygen-dependent degradation domain is identical in mammalian, Xenopus, and rainbow trout HIF-1alpha proteins, suggesting a high degree of evolutionary conservation in degradation of HIF-1alpha protein.
哺乳动物缺氧诱导因子-1（HIF-1）是一种异源二聚体转录因子，当细胞暴露于缺氧条件时，它可控制多种参与糖酵解、红细胞生成和血管生成的基因的诱导表达。迄今为止，尚未在鱼类中研究HIF-1α的表达和功能，而鱼类在其自然环境中会经历氧张力的大幅波动。通过电泳迁移率变动分析，我们已确定虹鳟鱼细胞中存在一种缺氧诱导因子。我们还克隆了虹鳟鱼HIF-1α的全长cDNA（3605个碱基对），其预测的蛋白质序列有766个氨基酸，与人和小鼠的HIF-1α有61%的相似性。在蛋白质印迹分析中，针对虹鳟鱼HIF-1α蛋白N端部分（氨基酸12 - 363）和C端部分（氨基酸330 - 730）的多克隆抗体可识别虹鳟鱼和奇努克鲑鱼的HIF-1α蛋白。此外, 人及小鼠的HIF-1α蛋白能被虹鳟鱼N端抗HIF-1α抗体识别，但不能被C端HIF-1α抗体识别。通过将虹鳟鱼和奇努克鲑鱼细胞在20%至0.2% O₂的不同氧浓度下孵育1小时，研究了HIF-1α的积累情况。HIF-1α蛋白的最大积累发生在5% O₂（38托）时，这是常氧动物静脉血的典型氧张力。在有无蛋白酶体抑制剂MG-132存在的情况下进行的蛋白质稳定性实验表明，该抑制剂能够稳定蛋白质，而该蛋白质在常氧和缺氧条件下通常都是通过蛋白酶体途径降解的。值得注意的是，氧依赖性降解结构域的缺氧反应元件在哺乳动物、非洲爪蟾和虹鳟鱼的HIF-1α蛋白中是相同的，这表明在HIF-1α蛋白的降解过程中存在高度的进化保守性。
