College of Science, National University of Defense Technology, Changsha, 410073, Hunan, People's Republic of China.
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, 27599, USA.
Cell Mol Life Sci. 2018 Dec;75(24):4629-4641. doi: 10.1007/s00018-018-2920-y. Epub 2018 Sep 27.
Two types of vertebrate cryptochromes (Crys) are currently recognized. Type 2 Crys function in the molecular circadian clock as light-independent transcriptional repressors. Type 4 Crys are a newly discovered group with unknown function, although they are flavoproteins, and therefore, may function as photoreceptors. It has been postulated that Crys function in light-dependent magnetoreception, which is thought to contribute towards homing and migratory behaviors. Here we have cloned and annotated the full-length pigeon ClCry1, ClCry2, and ClCry4 genes, and characterized the full-length proteins and several site-directed mutants to investigate the roles of these proteins. ClCry1 and ClCry2 are phylogenetically grouped as Type 2 Crys and thus are expected to be core components of the pigeon circadian clock. Interestingly, we find that ClCry4 is properly annotated as a Type 4 Cry. It appears that many birds possess a Type 4 Cry which, as in pigeon, is misannotated. Like the Type 2 Crys, ClCry4 is widespread in pigeon tissues. However, unlike the Type 2 Crys, ClCry4 is cytosolic, and purified ClCry4 possesses FAD cofactor, which confers characteristic UV-Vis spectra as well as two photochemical activities. We find that ClCry4 undergoes light-dependent conformational change, which is a property of insect Type 1 Crys involved in the insect-specific pathway of photoentrainment of the biological clock. ClCry4 can also be photochemically reduced by a mechanism common to all FAD-containing Cry family members, and this mechanism is postulated to be influenced by the geomagnetic field. Thus pigeon Crys control circadian behavior and may also have photosensory function.
目前已识别出两种脊椎动物隐花色素（Cry）。第二类 Cry 在分子生物钟中作为非依赖光照的转录抑制因子发挥作用。第四类 Cry 是新发现的一类，其功能未知，尽管它们是黄素蛋白，因此可能作为光受体发挥作用。有人假设 Cry 在依赖光照的磁感受中发挥作用，这被认为有助于归巢和迁徙行为。在这里，我们克隆并注释了全长鸽子 ClCry1、ClCry2 和 ClCry4 基因，并对全长蛋白和几个定点突变体进行了特征描述，以研究这些蛋白的作用。ClCry1 和 ClCry2 在系统发育上被归类为第二类 Cry，因此有望成为鸽子生物钟的核心成分。有趣的是，我们发现 ClCry4 被正确注释为第四类 Cry。似乎许多鸟类都拥有第四类 Cry，就像鸽子一样，它被错误注释了。与第二类 Cry 一样，ClCry4 在鸽子组织中广泛存在。然而，与第二类 Cry 不同的是，ClCry4 位于细胞质中，并且纯化的 ClCry4 具有 FAD 辅因子，赋予其特征性的 UV-Vis 光谱以及两种光化学活性。我们发现 ClCry4 会发生依赖光照的构象变化，这是一种参与生物钟昆虫特定光感应途径的昆虫类 1 Cry 的特性。ClCry4 还可以通过一种机制发生光化学还原，这种机制存在于所有含 FAD 的 Cry 家族成员中，并且该机制被推测受到地磁场的影响。因此，鸽子 Cry 控制昼夜节律行为，并且可能具有光感觉功能。
