Macromolécules Biologiques, Centre for Protein Engineering, Université de Liège, Liège, Belgium.
PLoS One. 2013 Sep 20;8(9):e68014. doi: 10.1371/journal.pone.0068014. eCollection 2013.
The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen.
大多数蛋白酶以无活性形式合成，称为酶原，由前肽和蛋白酶结构域组成。前肽通常参与酶的正确折叠和特异性抑制。屋尘螨过敏原 Der p 3 的前肽 NPILPASPNAT 含有一个脯氨酸丰富的基序（PRM），这对于胰蛋白酶样蛋白酶来说是不寻常的。通过截断前肽或用丙氨酸取代非糖基化酶原中的一个或所有脯氨酸，我们证明全长前肽对于正确折叠和热稳定性不是必需的，并且 PRM 对于在巴斯德毕赤酵母中表达时防止 proDer p 3 不受期望的蛋白水解至关重要。此外，我们通过将猝灭流动测定法与质谱分析相结合来跟踪 proDer p 3 的成熟时间过程。这种方法允许监测不同物种的演变，并确定主要过敏原 Der p 1 激活酶原的稳态动力学参数。该实验表明，脯氨酸 5 和 8 对于 proDer p 3-Der p 1 相互作用和酶原的激活至关重要。
