MAPPING THE STIMULUS-SPECIFIC SIGNALING PATHWAYS IN PERIODONTITIS BY PROTEOMICS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Periodontitis is an inflammatory disease with host-parasite interactions which contribute to connective tissue destruction and alveolar bone resorption. Porphyromonas gingivalis (P.g.) a black-pigmented Gram-negative anaerobic bacterium, is a major pathogen in the development and progression of periodontitis. Cell wall and appendage structures, such as lipopolysaccharide (LPS) and fimbriae, play important roles in the induction of innate immune responses, including cytokine production by localized and circulating monocyte/macrophage. Based on preliminary data, live P.g. stimulates unique pro-inflammatory signal transduction pathways in human peripheral blood monocytes (PBM) as opposed to bacterial components, such as LPS or fimbriae. In these experiments we test at the level of protein expression that unique signaling pathways are differentially induced by live P.g. The crude fimbriae are purified on a C8 reversed-phase column. (1) Human PBM (95% pure) freshly elutriated from healthy donor and monocyte-like THP-1 cells are exposed to live P.g., P.g. LPS and P.g. fimbriae. 2-dimensional gel electrophoresis (2-DGE) are performed on the cell lysate proteins of PBM and THP-1 differentially produced. The expressed proteins are excised and subjected to tryptic in-gel digestion. Matrix-assisted laser desorption/ionization (MALDI) spectra are acquired and protein identification is performed using online protein database search. P.g. crude fimbriae are well resolved from other components on a C8 column when eluted with a gradient of CH3CN in 0.1% TFA . By using RP-HPLC with C8 column, the P.g. fimbriae can be purified rapidly and efficiently. 2-DGE was performed on cell lysate proteins differentially produced by PBM when challenged by live P.g. vs. unchallenged conditions. The 2-DGE profiles identified several differentially expressed protein spots. To rule out protein spots resulting from P.g. proteins we performed also 2-DGE of P.g. and used it against the experimental gel. The differentially expressed proteins for P.g. challenged PBM were excised, in-gel digested with trypsin and mass spectra were obtained to characterize them. The results so far show that specific proteins are induced or down-regulated by live P.g. Similar procedures were implemented for LPS and fimbriae induced PBM. 1-D gel electrophoresis was performed on the PBM challenged by live P.g., LPS and fimbriae. This approach allowed for identification of biologically significant proteins that might be in low abundance. The separated protein bands were subjected to in-gel tryptic digestion and MALDI-MS spectra were obtained. On-line data base search was undertaken to identify the proteins. To improve separation during isoelectric focusing stage of 2-DGE, novel buffer conditions were used for extraction of proteins from the cell lysates of PBM and THP-1. The extraction buffer avoids the use of SDS, which hampers separation of proteins in the first dimension. The 2-DGE profiles showed similar patterns of protein separation for control, live P.g., LPS (Figure 3) and fimbriae induced THP-1, making it easier for identification of differentially expressed protein spots by direct visualization. Analysis of the 2-DGE profiles identified several differentially expressed protein spots. MALDI spectra were acquired and protein identification was performed using online protein database search. Similar approach will be undertaken for human PBM in the future. Post-translational modifications of the identified proteins will also be determined. 1. Sojar, H.T., Hamada, N. and Genco, R.J. Protein Expression and Purif. 9, 49-52 (1997).