Characterization of tissue transglutaminase in human osteoblast-like cells

Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5'-triph osphate (GTP) binding enzyme, which catalyzes the post-translational modifi cation of proteins by forming intermolecular epsilon(gamma -glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluati on of transglutaminase (TG) activity determined using the [1,4(14)C]-putres cine incorporation assay showed that the enzyme was active in all cell type s. However, there. was a significantly higher activity in the cell homogena tes of MG-63 cells as compared with HOB and HOS cells (p<0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation o f cells with tTG antibody, which led to inhibition of cell attachment. West ern blot analysis clearly indicated that the active TG was tTG and immunocy tochemistry showed it be situated in the cytosol of the cells. In situ extr acellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, w hich colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tT G to cell surface/matrix interactions and to the stabilization of the ECM o f osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.