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Role of γ-glutamyltranspeptidase in osteoclastogenesis induced by Fusobacterium nucleatum KCI 등재후보
We previously showed that γ-glutamyltranspeptidase (GGT), an enzyme involved in glutathione metabolism, in Bacillus subtilis acts as a virulence factor for osteoclastogenesis via the RANKL-dependent pathway. Hence, it can be hypothesized that GGT of periodontopathic bacteria acts as a virulence factor in bone destruction. Because Fusobacterium nucleatum, which is a periodontopathic pathogen, has GGT with a primary structure similar to that of B. subtilis GGT (37.7% identify), the bone-resorbing activity of F. nucleatum GGT was examined here. Recombinant GGT (rGGT) of F. nucleatum was expressed in Escherichia coli and purified using the His tag of rGGT. F. nucleatum rGGT (Fn rGGT) was expressed as a precursor of GGT, and then processed to a heavy subunit and a light subunit, which is characteristic of general GGTs, including the human and B. subtilis enzymes. Osteoclastogenesis was achieved in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. Fn rGGT induced osteoclastogenesis to a level similar to that of B. subtilis rGGT; furthermore, osteoclastogenesis was induced in a dose-dependent manner. These results suggest that F. nucleatum GGT possesses a virulent bone-resorbing activity, which could play an important role in the pathogenesis of periodontitis.
Materials and Methods
1. Alignment of amino acid sequences
2. Bacterial strains and culture media
3. Cloning of ggt gene
4. Expression and purification of Fn rGGT protein
5. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) analysis
6. Co-culture of osteoblast and bone marrow-derivedmacrophage
7. Tartrate resistant acid phosphatase (TRAP) staining
Results
1. Homology of GGT protein among F. nucleatum ,human and B. subtilis
2. Purification of F. nucleatum recombinant GGT
3. Effect of rGGT proteins on osteoclastogenesis
Discussion
References
