Ruggiero Sabrina, Cosgarea Raluca, Potempa Jan, Potempa Barbara, Eick Sigrun, Chiquet Matthias
Department of Orthodontics and Dentofacial Orthopedics, University of Bern, Switzerland.
Biochim Biophys Acta. 2013 Apr;1832(4):517-26. doi: 10.1016/j.bbadis.2013.01.003. Epub 2013 Jan 9.
Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease.
牙龈蛋白酶是半胱氨酸蛋白酶，是牙周致病细菌牙龈卟啉单胞菌的主要毒力因子。据报道，牙龈蛋白酶可降解牙周组织的细胞外基质（ECM），导致组织破坏和细胞凋亡。然而，确切机制尚不清楚。纤连蛋白和肌腱蛋白-C是牙周组织中存在的细胞周围ECM糖蛋白。纤连蛋白介导成纤维细胞黏附，而肌腱蛋白-C与纤连蛋白结合并抑制其细胞铺展活性。我们在体外使用纯化蛋白，研究了在临床相关浓度下纤连蛋白和肌腱蛋白-C是否会被牙龈蛋白酶切割，以及细菌蛋白酶切割如何影响它们在细胞黏附中的生物学活性。所有三种牙龈蛋白酶均可将纤连蛋白切割成不同片段；然而，只有精氨酸特异性的HRgpA和RgpB，而不是赖氨酸特异性的Kgp，破坏了其细胞铺展活性。纤连蛋白重组细胞结合结构域证实了这一结果。在两种主要的肌腱蛋白-C剪接变体中，大的变体而非小的变体是牙龈蛋白酶的底物，这表明切割主要发生在可变剪接结构域。令人惊讶的是，所有三种牙龈蛋白酶对大的肌腱蛋白-C变体的切割产生了对完整纤连蛋白具有增强抗黏附活性的片段。在一部分牙周炎患者的龈沟液中检测到了纤连蛋白和肌腱蛋白-C片段。我们得出结论，牙龈蛋白酶的切割直接影响纤连蛋白和肌腱蛋白-C的生物学活性，其方式可能导致牙周疾病期间细胞脱离增加和丢失。
