White, Peter Andrew; (1996) Bone-modulating proteins from Actinobacillus actinomycetemcomitans: Isolation, characterization and cloning. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus which is responsible for a number of severe infections, including localized juvenile periodontitis (LJP). Surface-associated material (SAM) from this organism, isolated by a gentle saline extraction, has the capacity to modulate the complex process of bone remodelling. Specifically SAM could inhibit mammalian cell proliferation including the bone cell population known as osteoblasts and could stimulate bone destruction in vitro. This investigation was concerned with the isolation, characterization and cloning of these bioactive components. SAM produced a dose-dependent inhibition of tritiated thymidine incorporation by numerous cell lines in culture, including the osteoblast-like cell line MG63. Characterization of the anti-proliferative activity in the SAM demonstrated that it was not cytotoxic and was heat- and trypsin-sensitive. A purification strategy was developed and the activity was fractionated using ammonium sulphate precipitation, anion exchange and size exclusion HPLC. Analysis of the active peak by SDS-PAGE and silver staining revealed a single protein with a molecular mass of approximately 8kDa. The mechanism by which this protein inhibits thymidine incorporation is unusual. Unlike direct inhibitors of DNA synthesis it has no effect on DNA, RNA or protein synthesis but acts by blocking mammalian cell cycle progression from the G2 to the M phase. The anti-proliferative protein from A. actinomycetemcomitans has been termed gapstatin. IgG antibodies to constituents of the SAM were found in the blood of patients with localized juvenile periodontitis (LJP). Sera from a proportion of patients with LJP significantly neutralized the anti-proliferative activity of the SAM, while control sera, from individuals with no evidence of periodontal disease, were unable to neutralize this activity. In order to clone the anti-proliferative protein, these sera were used to screen a genomic library of A. actinomycetemcomitans constructed by ligating Sau3A-digested and size- fractionated DNA into BamH1-cleaved pUC18. Although three clones expressing antigenic proteins were identified, no anti-proliferative activity was associated with them. SAM from A. actinomycetemcomitans was found to stimulate bone resorption as assessed using the mouse calvarial bone resorption assay. A monoclonal antibody to whole A. actinomycetemcomitans was developed and found to inhibit SAM-induced bone resorption. This information has facilitated the isolation and N-terminus sequencing of the bone resorbing protein in the SAM of A. actinomycetemcomitans, defining it as member of the heat-shock 60 family of proteins. Attempts were made to clone the gene expressing this protein.

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