Ishikawa Yoshihiro, Toups Melissa A, Elkrewi Marwan, Zajac Allison L, Horne-Badovinac Sally, Matsubayashi Yutaka
Department of Ophthalmology, University of California, San Francisco, School of Medicine, CA USA.
Department of Life and Environmental Sciences, Faculty of Science and Technology, Bournemouth University, UK.
bioRxiv. 2025 Aug 7:2025.08.05.668786. doi: 10.1101/2025.08.05.668786.
Collagens are fundamental components of extracellular matrices, requiring precise intracellular post-translational modifications for proper function. Among the modifications, prolyl 4-hydroxylation is critical to stabilise the collagen triple helix. In humans, this reaction is mediated by collagen prolyl 4-hydroxylases (P4Hs). While humans possess three genes encoding these enzymes (P4Hαs), harbour at least 26 candidates for collagen P4Hαs despite its simple genome, and it is poorly understood which of them are actually working on collagen in the fly. In this study, we addressed this question by carrying out thorough bioinformatic and biochemical analyses. We demonstrate that among the 26 potential collagen P4Hαs, PH4αEFB shares the highest homology with vertebrate collagen P4Hαs. Furthermore, while collagen P4Hs and their substrates must exist in the same cells, our transcriptomic analyses at the tissue and single cell levels showed a global co-expression of but not the other P4Hα-related genes with the collagen IV genes. Moreover, expression of during embryogenesis was found to precede that of collagen IV, presumably enabling efficient collagen modification by PH4αEFB. Finally, biochemical assays confirm that PH4αEFB binds collagen, supporting its direct role in collagen IV modification. Collectively, we identify PH4αEFB as the primary and potentially constitutive prolyl 4-hydroxylase responsible for collagen IV biosynthesis in . Our findings highlight the remarkably simple nature of collagen IV biosynthesis, which may serve as a blueprint for defining the minimal requirements for collagen engineering.
胶原蛋白是细胞外基质的基本成分，需要精确的细胞内翻译后修饰才能正常发挥功能。在这些修饰中，脯氨酰4-羟化对于稳定胶原蛋白三螺旋至关重要。在人类中，此反应由胶原蛋白脯氨酰4-羟化酶（P4Hs）介导。人类拥有三个编码这些酶的基因（P4Hαs），尽管其基因组简单，但果蝇中至少有26个胶原蛋白P4Hαs候选基因，而对于其中哪些基因实际上在果蝇体内作用于胶原蛋白，人们了解甚少。在本研究中，我们通过进行全面的生物信息学和生化分析来解决这个问题。我们证明，在26个潜在的胶原蛋白P4Hαs中，PH4αEFB与脊椎动物胶原蛋白P4Hαs具有最高的同源性。此外，虽然胶原蛋白P4Hs及其底物必须存在于同一细胞中，但我们在组织和单细胞水平的转录组分析表明，胶原蛋白IV基因与PH4αEFB共表达，而与其他P4Hα相关基因不共表达。此外，发现在胚胎发育过程中PH4αEFB的表达先于胶原蛋白IV的表达，推测这使得PH4αEFB能够有效地修饰胶原蛋白。最后，生化分析证实PH4αEFB与胶原蛋白结合，支持其在胶原蛋白IV修饰中的直接作用。总体而言，我们确定PH4αEFB是果蝇中负责胶原蛋白IV生物合成的主要且可能是组成型的脯氨酰4-羟化酶。我们的研究结果突出了果蝇胶原蛋白IV生物合成的显著简单性质，这可能为定义胶原蛋白工程的最低要求提供蓝图。
