Gingipains of Porphyromonas gingivalis Affect the Stability and Function of Serine Protease Inhibitor of Kazal-type 6 (SPINK6), a Tissue Inhibitor of Human Kallikreins

Abstract

Periodontitis, a chronic inflammation driven by dysbiotic subgingival bacterial flora, is linked on clinical levels to the development of a number of systemic diseases and to the development of oral and gastric tract tumors. A key pathogen, Porphyromonas gingivalis, secretes gingipains, cysteine proteases implicated as the main factors in the development of periodontitis. Here we hypothesize that gingipains may be linked to systemic pathologies through the deregulation of kallikrein-like proteinase (KLK) family members. KLKs are implicated in cancer development and are clinically utilized as tumor progression markers. In tissues, KLK activity is strictly controlled by a limited number of tissue-specific inhibitors, including SPINK6, an inhibitor of these proteases in skin and oral epithelium. Here we identify gingipains as the only P. gingivalis proteases responsible for SPINK6 degradation. We further show that gingipains, even at low nanomolar concentrations, cleaved SPINK6 in concentration-and time-dependent manner. The proteolysis was accompanied by loss of inhibition against KLK13. We also mapped the cleavage by Arg-specific gingipains to the reactive site loop of the SPINK6 inhibitor. Moreover, we identified a significant fraction of SPINK6-sensitive proteases in healthy saliva and confirmed the ability of gingipains to inactivate SPINK6 under ex vivo conditions. Finally, we demonstrate the double-edge action of gingipains, which, in addition, can activate KLKs because of gingipain K-mediated proteolytic processing of the zymogenic proform of KLK13. Altogether, the results indicate the potential of P. gingivalis to disrupt the control system of KLKs, providing a possible mechanistic link between periodontal disease and tumor development.

Kallikrein-related peptidase 14 activates zymogens of membrane type matrix metalloproteinases (MT-MMPs) - a CleavEx library-based analysis

“…Each native proMMP (0.5 µg) was incubated with 50 and 100 nM KLK14 (proMMP14) or 100 and 250 nM KLK14 (proMMP15, 16 and 17) for 1 hour at 37°C. After incubation, KLK14 was inhibited with 10 µM biotin-Tyr-Gly-Pro-Arg-CMK, a specific KLK14 inhibitor, and 1 µM serine protease inhibitor Kazal-type 6 (SPINK6) [66] for 15 minutes at 37°C. Next, non-reducing sample buffer (1:1) was added and samples were incubated for 30 minutes at 37°C.…”

“…5% CO2 to 80% confluency. The selection media was aspirated and cells were gently washed with PBS, then treated with 250 µl DMEM containing either 50 nM KLK14, 50 nM furin and each enzyme preincubated (15 min at 37°C) with its specific inhibitor: either 250 nM SPINK6[66] or 1 µM decanoyl-RVKR-CMK (Bachem), respectively and further incubated for 30 min at 37°C, 5% CO2.The supernatant was aspirated and the cells were rinsed with cold PBS three times and then treated with 0.1 mg/ml EZ-link TM Sulfo-NHS-LC-Biotin (Thermo Scientific) for 1 hour. Next, the cells were washed three times with PBS containing 100 mM glycine and then lysed with 500 µl RIPA buffer(10 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% DOC, 2 mM EDTA) containing 25X cOmplete TM EDTA-free Protease Inhibitor Cocktail (Roche) and 5 mM EDTA.…”

Proteases: Pivot Points in Functional Proteomics

“…Infectious organisms employ their own arsenal of proteases for propagation and virulence, such as HIV protease [175], Trypanosoma cruzi cruzipain [176], Porphyromonas gingivalis gingipains [177], and Bacillus anthracis lethal factor [178]. Several bacterial, viral, protozoan and fungal proteases trigger inflammation by activating the intrinsic coagulation pathway [102], or act as procoagulants by non-canonical, direct activation of prothrombin [179].…”

“…P. gingivalis gingipains are cysteine proteases associated with this type of chronic inflammation, and they are the only bacterial proteases that degrade SPINK6, a Kazal-type inhibitor of various human kallikreins in skin and oral epithelium. Loss of this proteolytic control has been suggested as a link between periodontal disease and tumor development [177]. …”

The role of kinases and phosphatases in the pathobiology of porphyromonas gingivalis.

“…These proteases have been shown to play roles in P. gingivalis invasion, biofilm formation, evasion of host immune response, amino acid uptake from host proteins and fimbriae maturation [70]. Recent studies have proposed these gingipains may be involved in various systemic pathologies [71]. P. gingivalis secretes 3 separate but related cysteine proteases.…”