The effect of bacterial γ-glutamyltranspeptidase on osteoclastogenesis

Abstract

[한글]

γ-glutamyltranspeptidase (GGT)는 새로운 골흡수 인자로서 최근에 쥐 T-lymphoma 세포의 cDNA library에서 클로닝되었다. GGT는 모든 생명체에 광범위하게 존재하며 특히 Bacillus subtilis GGT는 구조나 효소적 특성이 포유동물의 GGT와 매우 흡사해 B. subtilis GGT를 가지고 파골세포형성능을 실험하였다. 파골세포형성능은 두개골과 골수세포를 사용한 혼합배양을 이용하였고 tartrate-resistant acid phosphatase 염색으로 평가하였다. GGT 를 과발현하는 B. subtilis 배양액에서는 wild type이나 ggt-deleted B. subtilis 배양액보다 현저한 파골세포형성능이 나타났다. GGT가 어떤 기전에 의해 파골세포 분화에 관여하는지 알아보기 위해 B. subtilis GGT를 Escherichia coli에 클로닝하여 발현시키고 His-tag 정제 방법으로 다량의 정제된 GGT를 취하였다. 정제된 GGT를 쥐 두개골의 조골세포에 처리한 결과 receptor activator of nuclear factor-κB ligand, cyclooxygenase-2, heparin-binding epidermal growth factor-like growth factor, amphiregulin의 mRNA 발현이 증가된 반면, OPG의 발현에는 거의 영향을 미치지 않았다. 이상의 결과는 세균의 GGT도 RANKL과 관련된 경로를 통해 골흡수와 파골세포 분화에 관여함을 나타낸다.

[영문]A novel bone-resorbing factor, γ-glutamyltranpeptidase (GGT), was recently cloned from mouse T-lymphoma cell cDNA library. Since the GGT is widely distributed in living organisms and Bacillus subtilis GGT exhibits high similarity in its primary structure and enzymatic characteristics with the mammalian GGTs, the B. subtilis GGT was examined for the osteoclast formation. The osteoclast formation was performed in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells, and determined by tartrate resistant acid phosphatase staining. Conditioned medium from GGT-overproducing B. subtilis culture showed significantly higher activity of osteoclast formation when compared to those from wild-type and ggt-deleted B. subtilis culture. To understand the mechanism of how GGT induce osteoclastogenesis, B. subtilis GGT was cloned, expressed in Escherichia coli and purified using His-tag purification. The purified GGT stimulated mRNA expression of the receptor activator of nuclear factor-κB ligand, cyclooxygenase-2, heparin-binding epidermal growth factor-like growth factor, and amphiregulin in mouse calvaria-derived osteoblasts.This report is the first demonstration that bacterial GGT is able to serve as the bone-resorbing factor and the osteoclastogenesis by the GGT is mediated by a RANKL-dependent pathway. Based on this study, it can be hypothesized that GGT in periodontopathic bacteria can be an important virulence factor for bone destruction in periodontitis.