An osteonectin-like protein in the matrix of cultured osteogenic cell-line MC3T3-E1, which is associated with calcification

Abstract

Time-dependent changes of the [3H]-proline-labeled noncollagenous proteins synthesized by the osteogenic cell-line MC3T3-E1 were analyzed over a range starting from cell confluency to 13 days after confluency during which time cells formed a bone-like structure. It was found that the 40 kDa protein on SDS-polyacrylamide gel (SDS-PAGE) remarkably increased in the cell-matrix layer at about 9 days after cell confluence, just before calcification. This protein was highly purified and was found to contain high amounts of glutamic acid, glycine, and serine. An internal amino acid sequence of this protein was revealed to be K-X-M-A-P-E-E-X-P, which showed homology with the sequence of the EF-hand domain in osteonectin/SPARC (secreted protein, acidic, and rich in cysteine). This protein co-migrated with collagen in gel filtration, and ion-exchange chromatography. Furthermore, it showed high affinity to type I collagen.