While the antimicrobial peptide LL-37 was found to play a crucial role in preventing periodontal disease associated with Actinobacillus actinomycetemcomitans, later works reported the resistance of Porphyromonas gingivalis to LL-37. This periopathogen is known for its high proteolytic activity related to its gingipains proteases. Objectives: Our goal is to investigate the ability of proteases from P. gingivalis to hydrolyze LL-37 under in-vitro and ex-vivo conditions. Methods: LL-37 integrity was detected by western blot analysis following its incubation with supernatant from P. gingivalis. ARG-gingipain activity was measured on a chromogenic substrate. Saliva was collected from healthy volunteers. Binding of LL-37 to salivary components was demonstrated by Far-western analysis and the salivary glycoproteins were detected by a glycoprotein detection kit. Results: Under in-vitro conditions, we found that proteases from P. gingivalis can degrade LL-37. By using supernatant from gingipains mutant strains we revealed that the P. gingivalis Arg-gingipain digests LL-37. Surprisingly, the presence of saliva prevented the degradation of LL-37 and remained the peptide intact (under ex-vivo conditions). BSA at higher concentration than that of salivary proteins did not prevent LL-37 degradation. LL-37 protection from degradation was heat resistant as it was preserved even when boiled saliva was used. Saliva did not inhibit the activity of ARG-gingipain. Saliva did not prevent the degradation of a short protein that was randomly chosen as control (hsp65). Incubation of LL-37 with saliva altered the mobility of the peptide in native gel. Far-western analysis demonstrated the binding of salivary component with high molecular weight to LL-37. Salivary proteins with identical molecular weight were identified as glycoproteins. Conclusions: Salivary component protects LL-37 from degradation in a specific mechanism. This component is most likely a high molecular weight glycoprotein.
This work was supported by the Israel Science Foundation grant number 517/06 and the Abisch-Frenkel foundation.