Cleavage of extracellular matrix in periodontitis: Gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C

Abstract

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.

Tenascin-C: Form versus function

“…Fragments of tenascin-C have been reported in swabs from individuals with gingivitis, 189 chronic venous leg ulcer exudates, 164 and atherosclerotic plaques 190 among other locations.…”

Tenascin-C at a glance

“…Finally, tenascin-C can be cleaved by matrix metalloproteases (MMPs) (Giblin and Midwood, 2015) and gingipain cysteine proteases (Ruggiero et al, 2013), which contributes to tenascin-C turnover in tissue. In vitro cleavage of purified tenascin-C through MMP-1,-2, -3 and -7 demonstrated that the majority of cleavage sites are located within the alternatively spliced domains, although MMP-7 and the proteinase ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) both also cleave within the assembly domain.…”

Porphyromonas gingivalis gingipains induce a pro-inflammatory extracellular microenvironment : the role of PAR-2 and fibronectin.

“…This differential affinity towards fibronectin is most likely due to the presence of the hemaglutinin domains in the HRgpA complex, which bind fibronectin facilitating proteolytic fragmentation. Our results of HRgpA dependent fibronectin fragmentation are consistent with the results of Ruggiero and others [108]. The presence of the hemaglutinin adhesion domain may serve as an important anchoring device to the extracellular milieu within the periodontal pocket.…”

“…P. gingivalis use its gingipains to proteolytically cleave integrin-gingipains interactions on fibroblast cell surface [107]. Previous studies demonstrate that gingival fibroblasts when cocultured with a high concentration of purified gingipains lead to a decrease in fibronectin and α5β1 integrin, causing cell detachment and cell death [108]. Not surprisingly, samples taken from patients with periodontitis demonstrated increased amounts of fibronectin fragments, the result of host and pathogen proteolysis [109,110].…”