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Abstract：
The extended cleavage specificities of two hematopoietic serine proteases originating from the ray-finned fish, the spotted gar (Lepisosteus oculatus), 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.
摘要：
来自射线鳍鱼的两种造血丝氨酸蛋白酶的扩展切割特异性，斑点gar（Lepisosteusoculatus），已经使用底物噬菌体展示进行了表征。使用一组重组底物进一步验证切割位点处和周围的特定氨基酸的偏好。对于其中一种酶，gar颗粒酶G，在可切割的P1位置观察到对芳香族氨基酸Tyr的严格优选。使用一组重组底物表明，gar粒酶G对Tyr具有很高的选择性，但在Phe后而不是Trp后对裂解的活性较低。相反，第二种酶，garDDN1在底物的P1位置显示出对Leu的高度偏好。后一种酶也显示出在P2位置对Pro和在P4和P5位置对Arg的高度偏好。P4和P5位的两个Arg残基的选择性表明该酶具有高度特异性的底物选择性。用底物噬菌体展示获得的这两种蛋白酶的共有序列筛选gar蛋白质组产生了非常不同的潜在靶标。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其组织分布。幼鱼的组织切片显示两种蛋白酶在肠道区域Peyer的斑块样结构中的细胞中表达，表明它们可能在T或NK细胞中表达。然而,由于缺乏针对gar中特定表面标记的抗体，还无法确定确切的细胞来源。观察到两种蛋白酶的丰度存在显着差异，其中garDDN1的表达水平高于gar颗粒酶G。两者似乎都在相同或相似的细胞中表达，具有类似淋巴细胞的外观。
