Department of Anatomy and Molecular Embryology, Medical Faculty, Ruhr-University Bochum, Bochum, Germany.
Molecular and Experimental Cardiology, St. Josef-Hospital, Clinics of the Ruhr University Bochum, Bochum, Germany.
PLoS One. 2021 Jul 30;16(7):e0253476. doi: 10.1371/journal.pone.0253476. eCollection 2021.
Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris. Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions.
脱氧核糖核酸酶 1（DNase1）家族的可溶性核酸酶促进某些形式的细胞分化和死亡过程中的 DNA 和染色质的处理（染色质溶解），并参与抑制抗核自身免疫以及由聚集的中性粒细胞胞外陷阱引起的血栓性微血管病。由于对分泌型 DNase1 家族成员的特异性活性和特性仍然缺乏系统和直接的比较，我们使用巴斯德毕赤酵母表达和纯化重组鼠 DNase1（rmDNase1）、DNase1 样蛋白 2（rmDNase1L2）和 DNase1 样蛋白 3（rmDNase1L3）。采用不同的策略优化培养和纯化条件，我们在表达介质中获得了纯度蛋白的产量在3mg/L（rmDNase1L2 和 rmDNase1L3）和9mg/L（rmDNase1）之间。此外，我们建立了一种对仍与酵母α交配因子的未加工三 N-糖基化前肽结合的不成熟 DNase 进行翻译后成熟的程序。我们通过超增色测定分析了糖基化谱，并测定了特定的 DNase 活性。此外，我们通过在有或没有肝素和单体骨骼肌α-肌动蛋白的情况下，使用 lambda DNA 和染色质消化测定，在等摩尔 DNase 同工型浓度下，在各种条件下评估了底物特异性。我们的结果表明，由于其生化特性，mDNase1L2 可以被视为 mDNase1 和 mDNase1L3 的进化中间同工型。因此，我们的数据表明，在广泛的生物学条件下，分泌型 DNase1 家族成员相互补充，以实现最佳的 DNA 降解和染色质溶解。
