CHARACTERIZATION OF THE OSTEOGENIC STROMAL CELL-LINE MN7 - IDENTIFICATION OF SECRETED MN7 PROTEINS USING 2-DIMENSIONAL POLYACRYLAMIDE-GEL ELECTROPHORESIS, WESTERN BLOTTING, AND MICROSEQUENCING

Proteins secreted by the osteogenic stromal cell line MN7 were analyze d using two-dimensional polyacrylamide gel electrophoresis (PAGE), wes tern blotting, immunodetection, and microsequencing. Trichloroacetic a cid-precipitated proteins from the conditioned medium of MN7 cell cult ures, harvested at different times of growth, were dissolved in denatu ring and reducing sample buffer and separated in the first dimension a ccording to isoelectric point and in the second dimension according to molecular weight. Protein patterns were visualized using silver stain ing. Among the 350 separated protein spots, we identified type I colla gen, bone sialoprotein, osteonectin, and cathepsin B by western blotti ng and immunodetection using polyclonal antibodies. Osteocalcin could not be detected in the conditioned medium of MN7 cells. Furthermore, 1 5 MN7-specific protein spots were localized after comparison with two- dimensional PAGE patterns from the conditioned medium of the nonosteog enic stromal cell lines MM1 and MV1. Microsequencing of the internal p eptides of five selected spots revealed three known proteins, namely t he carboxyl-terminal propeptide of the a, chain of collagen type I, ca thepsin L, and the tissue inhibitor of metalloproteinases-2, an 18 kil odalton peptide fragment from osteopontin that has not previously been described, and a novel glycosylated 85 kD protein with an average iso electric point of 5.7. All identified proteins did not vary in presenc e between the different time points analyzed by two-dimensional PAGE. The use of two-dimensional PAGE to investigate the secreted proteins o f MN7 cells will enable us to establish a complete protein data base o f extracellular osteoblast-specific proteins. Furthermore, two-dimensi onal PAGE in combination with other techniques is a fast and accurate method for the identification of novel proteins that could function as markers in osteoblast differentiation and/or bone formation.