Department of Cell and Molecular Biology, Uppsala University, The Biomedical Center, Box 596, SE-751 24, Uppsala, Sweden.
Department of Cell and Molecular Biology, Uppsala University, The Biomedical Center, Box 596, SE-751 24, Uppsala, Sweden; Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Box 7011, SE-75007, Uppsala, Sweden.
Dev Comp Immunol. 2023 Nov;148:104920. doi: 10.1016/j.dci.2023.104920. Epub 2023 Aug 18.
Granzymes A and K are two highly homologous serine proteases expressed by mammalian cytotoxic T cells (CTLs) and natural killer (NK) cells. The locus encoding these two proteases is the first of the hematopoietic serine protease loci to appear during vertebrate evolution. This locus is found in all jawed vertebrates including the cartilaginous fishes. Granzyme A is the most abundant of the different granzymes expressed by CTLs and NK cells and its potential function has been studied extensively for many years. However, no clear conclusions concerning its primary role in the immune defense has been obtained. In all mammals, there are only one copy each of granzyme A and K, whereas additional copies are found in both cartilaginous and ray finned fishes. In cichlids two of these copies seem to encode new members of the granzyme A/K family. These two new members appear to have changed primary specificity and to be pure chymases based on the amino acids in their active site substrate binding pockets. Interestingly, one of these gene copies is located in the middle of the granzyme A/K locus, while the other copy is present in another locus, the met-ase locus. We here present a detailed characterization of the extended cleavage specificity of one of these non-classical granzymes, a Zebra mbuna granzyme positioned in the granzyme A/K locus. This enzyme, named granzyme A2, showed a high preference for tyrosine in the P1 position of substrates, thereby being a strict chymase. We have also characterized one of the classical granzyme A/Ks of the Zebra mbuna, granzyme A1, which is a tryptase with preference for arginine in the P1 position of substrates. Based on their extended specificities, the two granzymes showed major similarities, but also some differences in preferred amino acids in positions surrounding the cleavable amino acid. Fish lack one of the hematopoietic serine protease loci of mammals, the chymase locus, where one of the major mast cell enzymes is located. An interesting question is now if cichlids have by compensatory mechanisms generated a mast cell chymase from another locus, and if similar chymotryptic enzymes have appeared also in other fish species.
颗粒酶 A 和 K 是两种高度同源的丝氨酸蛋白酶，由哺乳动物细胞毒性 T 细胞 (CTL) 和自然杀伤 (NK) 细胞表达。编码这两种蛋白酶的基因座是脊椎动物进化过程中第一个出现的造血丝氨酸蛋白酶基因座。该基因座存在于所有有颌脊椎动物中，包括软骨鱼类。颗粒酶 A 是 CTL 和 NK 细胞表达的不同颗粒酶中最丰富的一种，其潜在功能已被广泛研究多年。然而，对于其在免疫防御中的主要作用，尚未得出明确的结论。在所有哺乳动物中，颗粒酶 A 和 K 各只有一个拷贝，而软骨鱼类和硬骨鱼类中则发现了额外的拷贝。在慈鲷鱼中，其中两个拷贝似乎编码了颗粒酶 A/K 家族的新成员。这两个新成员似乎改变了主要特异性，并且基于其活性位点底物结合口袋中的氨基酸，它们是纯糜酶。有趣的是，这些基因拷贝中的一个位于颗粒酶 A/K 基因座的中间，而另一个拷贝存在于另一个基因座，即 met-ase 基因座。我们在这里详细描述了位于颗粒酶 A/K 基因座中的一个非经典颗粒酶，即斑马雀鲷颗粒酶 A2 的扩展切割特异性。这种酶被命名为颗粒酶 A2，它对底物 P1 位的酪氨酸表现出高偏好性，因此是一种严格的糜酶。我们还对斑马雀鲷的一种经典颗粒酶 A/K，即颗粒酶 A1 进行了表征，它是一种对底物 P1 位的精氨酸具有偏好性的胰凝乳蛋白酶。基于它们的扩展特异性，这两种颗粒酶表现出主要的相似性，但在切割氨基酸周围的位置上的偏好氨基酸方面也存在一些差异。鱼类缺乏哺乳动物造血丝氨酸蛋白酶基因座之一，即糜酶基因座，其中一种主要的肥大细胞酶就位于该基因座。一个有趣的问题是，慈鲷鱼是否通过补偿机制从另一个基因座产生了一种肥大细胞糜酶，以及类似的糜蛋白酶是否也出现在其他鱼类中。
