Recombinant production and characterization of the human mast cell tryptases gamma and delta

胰蛋白酶是丝氨酸蛋白酶，是肥大细胞中最丰富和最特异的蛋白质之一。在人类中已经发现了四种胰蛋白酶，但只有胰蛋白酶α和β在功能和结构上得到了很好的表征，这表明β在肥大细胞与疾病相关的“坏”和最近发现的维持健康的“好”功能中都是一个关键的中介。相比之下，胰蛋白酶γ和δ很少受到关注，并且在它们的功能和它们对肥大细胞生物学的贡献方面仍然是孤儿。在这个项目中，我们已经在酵母和哺乳动物细胞中表达了人类胰蛋白酶γ和δ及其变体，从而在我们的实验室中获得了所有人类胰蛋白酶。我们目前正在使用这些重组胰蛋白酶来研究它们的酶活性和调控，阐明它们的结构，并产生检测和抑制它们的工具。我们计划专门利用这些工具来探索膜结合胰蛋白酶γ的显著特征及其对胰蛋白酶中介作用的贡献。我们认为，理解和分离单个胰蛋白酶的特性和功能对于理解胰蛋白酶作为一个群体以及使用任何胰蛋白酶作为靶点的治疗干预至关重要。

Tryptases are serine proteinases that are among the most abundant and specific proteins of mast cells. Four tryptases have been identified in man, but only tryptase α and β have been functionally and structurally well characterized, showing that β is a key mediator involved in both the disease-related ‘bad’ and the more recently discovered healthmaintaining ‘good’ functions of mast cells. In contrast, tryptase γ and δ have received little attention and have remained orphan in respect to their functions and their contributions to mast cell biology. In this project we have expressed human tryptase γ and δ and variants in yeast and mammalian cells, thereby making all human tryptases available in our lab. We are currently using these recombinant tryptases to study their enzymatic activity and regulation, to elucidate their structures and to generate tools for their detection and inhibition. We plan to employ these tools specifically to explore the distinguishing features of the membrane-bound tryptase γ and its contributions to the mediator actions of tryptases. We believe that understanding and separating the properties and functions of the individual tryptases is essential for the understanding of tryptases as a group and for therapeutic interventions using any tryptase as target.