Fu Zhirong, Akula Srinivas, Olsson Anna-Karin, Hellman Lars
Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
Department of Medical Biochemistry and Microbiology, BMC, Box 589, SE-751 23, Uppsala, Sweden.
Dev Comp Immunol. 2022 Apr;129:104337. doi: 10.1016/j.dci.2021.104337. Epub 2021 Dec 15.
Serine proteases are major granule constituents of cells from several mammalian hematopoietic cell lineages. Despite the relatively extensive knowledge about these mammalian proteases, very little is known about their bird, reptile and amphibian homologs. In order to close this gap in our understanding of the evolution of these proteases, we have characterized the extended cleavage specificity and hematopoietic expression pattern of the chicken serine protease cathepsin G-like. This protease, which clusters in a separate subfamily of serine proteases among the vertebrate hematopoietic serine proteases, has been characterized using substrate phage display and further validated by using a panel of recombinant substrates. A preference for a lysine in the P1 position of a substrate, arginines in positions P2 and P3, and the aromatic amino acid tryptophane in the P4 position was observed. Based on the sequence alignment we could identify a consensus sequence for this protease as being PGGWRRKALSV. Mass spectrometry analysis of a peptide with the consensus sequence obtained by phage display showed that cleavage of this peptide occurred after the conserved Lys (K) residue. A screening of potential in vivo substrates based on the derived P5-P3' consensus sequence resulted in a relatively limited number of potential substrates, due to the high selectivity of this enzyme. The most interesting of these were PDGF-A, coagulation factor V and low-density lipoprotein receptor like-8. Immunohistochemical analysis of chicken white blood cells with antisera produced against chicken cathepsin G-like and chicken egg lysozyme, as a reference protein known to be expressed by hematopoietic cells, showed presence of chicken cathepsin G-like almost exclusively in thrombocytes whereas lysozyme was found at very high amounts in heterophils, and lower amounts in monocytes and thrombocytes.
丝氨酸蛋白酶是几种哺乳动物造血细胞谱系细胞的主要颗粒成分。尽管对这些哺乳动物蛋白酶已有相对广泛的了解，但对它们在鸟类、爬行动物和两栖动物中的同源物却知之甚少。为了填补我们在理解这些蛋白酶进化方面的这一空白，我们对鸡丝氨酸蛋白酶组织蛋白酶G样的扩展切割特异性和造血表达模式进行了表征。这种蛋白酶在脊椎动物造血丝氨酸蛋白酶中聚集在丝氨酸蛋白酶的一个单独亚家族中，已通过底物噬菌体展示进行了表征，并通过一组重组底物进一步验证。观察到该蛋白酶对底物P1位置的赖氨酸、P2和P3位置的精氨酸以及P4位置的芳香族氨基酸色氨酸有偏好。基于序列比对，我们可以确定该蛋白酶的共有序列为PGGWRRKALSV。对通过噬菌体展示获得的具有共有序列的肽段进行质谱分析表明，该肽段的切割发生在保守的赖氨酸（K）残基之后。基于推导的P5-P3'共有序列对潜在体内底物进行筛选，由于该酶的高选择性，导致潜在底物数量相对有限。其中最有趣的是血小板衍生生长因子A、凝血因子V和低密度脂蛋白受体样8。用针对鸡组织蛋白酶G样产生的抗血清和作为已知由造血细胞表达的参考蛋白的鸡卵溶菌酶对鸡白细胞进行免疫组织化学分析，结果表明鸡组织蛋白酶G样几乎仅存在于血小板中，而溶菌酶在异嗜性粒细胞中含量很高，在单核细胞和血小板中含量较低。
