PDF(Pubmed)
Abstract：
Hydroxylation of prolines to 4-trans-hydroxyproline (Hyp) is mediated by prolyl-4 hydroxylases (P4Hs). In plants, Hyps occur in Hydroxyproline-rich glycoproteins (HRGPs), and are frequently O-glycosylated. While both modifications are important, e.g. for cell wall stability, they are undesired in plant-made pharmaceuticals. Sequence motifs for prolyl-hydroxylation were proposed but did not include data from mosses, such as Physcomitrella. We identified six moss P4Hs by phylogenetic reconstruction. Our analysis of 73 Hyps in 24 secretory proteins from multiple mass spectrometry datasets revealed that prolines near other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95 % of Hyps were predictable with combined established methods. In our data, AOV was the most frequent pattern. A combination of 443 AlphaFold models and MS data with 3000 prolines found Hyps mainly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a p4h1 knock-out (KO) Physcomitrella mutant. Quantitative proteomics with different p4h mutants revealed specific changes in protein amounts, and a modified prolyl-hydroxylation pattern, suggesting a differential function of the Physcomitrella P4Hs. Quantitative RT-PCR revealed a differential effect of single p4h KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 suggested significant amino acids in the active centre of the enzyme and revealed differences between P4H1 and the other Physcomitrella P4Hs.
摘要：
脯氨酸向4-反式-羟脯氨酸(Hyp)的羟化是由脯氨酸-4羟化酶(P4Hs)介导的。在植物中,Hyps发生在富含羟脯氨酸的糖蛋白（HRGP）中，并且经常是O-糖基化的。虽然两种修改都很重要，例如，为了细胞壁的稳定性，它们在植物制造的药物中是不受欢迎的。prolyl-羟基化的序列基序被提出，但不包括来自苔藓的数据，比如Physcomitrella.我们通过系统发育重建鉴定了六个苔藓P4Hs。我们对来自多个质谱数据集的24种分泌蛋白中的73种Hyps的分析显示，脯氨酸接近其他脯氨酸，丙氨酸,丝氨酸,苏氨酸和缬氨酸优先羟基化。大约95%的Hyps是通过组合的既定方法可预测的。在我们的数据中,AOV是最常见的模式。443AlphaFold模型和MS数据与3000脯氨酸的组合发现Hyps主要在无序区域的蛋白质表面上。苔藓产生的人促红细胞生成素（EPO）在两个Hyps上显示出阿拉伯糖链的O-糖基化。这种修饰在p4h1敲除(KO)Physcomitrella突变体中显著减少。具有不同p4h突变体的定量蛋白质组学揭示了蛋白质数量的特定变化，和改良的脯氨酸-羟基化模式，表明PhyscomitrellaP4Hs的微分功能。定量RT-PCR揭示了单个p4hKOs对其他5个p4h基因表达的差异效应,暗示了对突变的部分补偿。PhyscomitrellaP4H1及其靶EPO肽的AlphaFold-Multimer模型与衣藻P4H1的晶体结构重叠，表明该酶的活性中心有重要的氨基酸，并揭示了P4H1与其他PhyscomitrellaP4Hs之间的差异。
