Characterization of tissue transglutaminase in human osteoblast-like cells.
Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5'-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification 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 evaluation of transglutaminase (TG) activity determined using the [1,4 14C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates 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 of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular 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, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.
Heath
D J
DJ
School of Biomedical Sciences, Queen's Medical Center, University of Nottingham, United Kingdom.
Downes
S
S
Verderio
E
E
Griffin
M
M
D015153:Blotting, Western; D002103:Cadaverine / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D005110:Extracellular Space / Q000378:metabolism; D005270:Femur Head; D005353:Fibronectins / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D010006:Osteoblasts / Q000166:cytology / Q000201:enzymology*; D012516:Osteosarcoma; D011503:Transglutaminases / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D005353:Fibronectins; D005452:Fluoresceins; D011503:Transglutaminases; D002103:Cadaverine