Department of Biosciences, University of Salzburg, Hellbrunner Straße 34, 5020, Salzburg, Austria.
Proteolysis Lab, Department of Structural Biology, Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, Baldiri Reixac, 15-21, 08028, Barcelona, Catalonia, Spain.
Sci Rep. 2020 Nov 4;10(1):19052. doi: 10.1038/s41598-020-76010-8.
Flagellins are the protein components of bacterial flagella and assemble in up to 20,000 copies to form extracellular flagellar filaments. An unusual family of flagellins was recently discovered that contains a unique metalloprotease domain within its surface-exposed hypervariable region. To date, these proteolytic flagellins (also termed flagellinolysins) have only been characterized in the Gram-positive organism Clostridium haemolyticum, where flagellinolysin was shown to be proteolytically active and capable of cleaving extracellular protein substrates. The biological function of flagellinolysin and its activity in other organisms, however, remain unclear. Here, using molecular biochemistry and proteomics, we have performed an initial characterization of a novel flagellinolysin identified from Hylemonella gracilis, a Gram-negative organism originally isolated from pond water. We demonstrate that H. gracilis flagellinolysin (HgrFlaMP) is an active calcium-dependent zinc metallopeptidase and characterize its cleavage specificity profile using both trypsin and GluC-derived peptide libraries and protein substrates. Based on high-throughput degradomic assays, HgrFlaMP cleaved 784 unique peptides and displayed a cleavage site specificity similar to flagellinolysin from C. haemolyticum. Additionally, by using a set of six protein substrates, we identified 206 protein-embedded cleavage sites, further refining the substrate preference of HgrFlaMP, which is dominated by large hydrophobic amino acids in P1', and small hydrophobic or medium-sized polar residues on the amino-terminal side of the scissile bond. Intriguingly, recombinant HgrFlaMP was also capable of cleaving full-length flagellins from another species, suggesting its potential involvement in interbacterial interactions. Our study reports the first experimentally characterized proteolytic flagellin in a Gram-negative organism, and provides new insights into flagellum-mediated enzymatic activity.
鞭毛蛋白是细菌鞭毛的蛋白质组成部分，可组装成多达 20000 个拷贝，形成细胞外鞭毛丝。最近发现了一种不寻常的鞭毛蛋白家族，其表面暴露的高变区含有独特的金属蛋白酶结构域。迄今为止，这些蛋白水解鞭毛蛋白（也称为鞭毛溶菌素）仅在革兰氏阳性菌溶血性梭菌中得到了表征，其中鞭毛溶菌素被证明具有蛋白水解活性，并能够切割细胞外蛋白底物。然而，鞭毛溶菌素的生物学功能及其在其他生物体中的活性仍不清楚。在这里，我们使用分子生化和蛋白质组学技术，对从池塘水中分离出的革兰氏阴性菌 gracilis 中鉴定出的一种新型鞭毛溶菌素进行了初步表征。我们证明，Hylemonella gracilis 鞭毛溶菌素（HgrFlaMP）是一种活性依赖于钙的锌金属肽酶，并使用胰蛋白酶和 GluC 衍生肽文库和蛋白质底物对其切割特异性进行了表征。基于高通量降解组学测定，HgrFlaMP 切割了 784 个独特的肽段，并显示出与溶血性梭菌鞭毛溶菌素相似的切割特异性。此外，通过使用一组 6 种蛋白质底物，我们鉴定了 206 个蛋白质嵌入的切割位点，进一步细化了 HgrFlaMP 的底物偏好性，其主要由 P1' 中的大疏水性氨基酸和靠近切割键的氨基端侧的小疏水性或中到大极性残基组成。有趣的是，重组 HgrFlaMP 还能够切割来自另一种物种的全长鞭毛蛋白，这表明它可能参与了细菌间的相互作用。我们的研究报告了革兰氏阴性菌中第一个经过实验表征的蛋白水解鞭毛蛋白，并为鞭毛介导的酶活性提供了新的见解。
