Department of Cell and Molecular Biology, Uppsala University, P.O. Box 596, SE-751 24 Uppsala, Sweden.
Department of Medical Biochemistry and Microbiology, Uppsala University Biomedical Centre (BMC), P.O. Box 582, SE-751 23 Uppsala, Sweden.
Int J Mol Sci. 2024 Jan 30;25(3):1669. doi: 10.3390/ijms25031669.
The extended cleavage specificities of two hematopoietic serine proteases originating from the ray-finned fish, the spotted gar (), have been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated using a panel of recombinant substrates. For one of the enzymes, the gar granzyme G, a strict preference for the aromatic amino acid Tyr was observed at the cleavable P1 position. Using a set of recombinant substrates showed that the gar granzyme G had a high selectivity for Tyr but a lower activity for cleaving after Phe but not after Trp. Instead, the second enzyme, gar DDN1, showed a high preference for Leu in the P1 position of substrates. This latter enzyme also showed a high preference for Pro in the P2 position and Arg in both P4 and P5 positions. The selectivity for the two Arg residues in positions P4 and P5 suggests a highly specific substrate selectivity of this enzyme. The screening of the gar proteome with the consensus sequences obtained by substrate phage display for these two proteases resulted in a very diverse set of potential targets. Due to this diversity, a clear candidate for a specific immune function of these two enzymes cannot yet be identified. Antisera developed against the recombinant gar enzymes were used to study their tissue distribution. Tissue sections from juvenile fish showed the expression of both proteases in cells in Peyer's patch-like structures in the intestinal region, indicating they may be expressed in T or NK cells. However, due to the lack of antibodies to specific surface markers in the gar, it has not been possible to specify the exact cellular origin. A marked difference in abundance was observed for the two proteases where gar DDN1 was expressed at higher levels than gar granzyme G. However, both appear to be expressed in the same or similar cells, having a lymphocyte-like appearance.
两种来源于硬骨鱼斑点叉尾鮰的造血丝氨酸蛋白酶（gar 颗粒酶 G 和 gar DDN1）的延伸裂解特异性已通过噬菌体展示底物进行了表征。使用一组重组底物进一步验证了裂解位点处和周围特定氨基酸的偏好性。对于其中一种酶 gar 颗粒酶 G，在可裂解的 P1 位置观察到对芳香族氨基酸 Tyr 的严格偏好。使用一组重组底物表明 gar 颗粒酶 G 对 Tyr 具有高选择性，但对 Phe 后而非 Trp 后切割的活性较低。相反，第二种酶 gar DDN1 在底物的 P1 位置显示出对 Leu 的高偏好性。这种酶还显示出对 P2 位置 Pro 和 P4 和 P5 位置 Arg 的高偏好性。对 P4 和 P5 位置的两个 Arg 残基的选择性表明该酶具有高度特异性的底物选择性。用这两种蛋白酶的底物噬菌体展示获得的共有序列筛选 gar 蛋白质组，得到了一组非常多样化的潜在靶标。由于这种多样性，尚不能确定这两种酶的特定免疫功能的明确候选物。针对重组 gar 酶开发的抗血清用于研究它们的组织分布。幼鱼组织切片显示这两种蛋白酶在肠道区域派伊尔氏斑样结构的细胞中表达，表明它们可能在 T 或 NK 细胞中表达。然而，由于 gar 中缺乏针对特定表面标志物的抗体，因此无法确定确切的细胞来源。观察到这两种蛋白酶的丰度存在明显差异，其中 gar DDN1 的表达水平高于 gar 颗粒酶 G。然而，两者似乎都在相同或相似的细胞中表达，具有淋巴细胞样外观。
