Functional analysis of bone sialoprotein: Identification of the hydroxyapatite-nucleating and cell-binding domains by recombinant peptide expression and site-directed mutagenesis

Mammalian bone sialoprotein (BSP) is a mineralized tissue-specific protein containing an RGD (arginine-glycine-aspartic acid) cell-attachment sequence and two distinct glutamic acid (glu)-rich regions, with each containing on e contiguous glu sequence. These regions have been proposed to contribute t o the attachment of bone cells to the extracellular matrix and to the nucle ation of hydroxyapatite (HA), respectively. To further delineate the domain s responsible for these activities, porcine BSP cDNA was used to construct expression vectors coding for two partial-length recombinant BSP peptides: P2S (residues 42-87), containing the first glutamic acid-rich domain; and P 1L (residues 69-300), containing the second glutamic acid-rich region and t he RGD sequence. These peptides were expressed in Escherichia coli as his-t ag fusion proteins and purified by nickel affinity columns and FPLC chromat ography, Digestion with trypsin released the his-tag fusion peptide, which generated P2S-TY (residues 42-87) and P1L-TY (residues 132-239), Using a st eady-state agarose gel system, P2S-TY promoted HA nucleation, whereas P2S, P1L, and P1L-TY did not. This implies that the minimum requirement for nucl eation of HA resides within the amino acid sequence of the first glutamic a cid-rich domain, whereas the second glutamic acid-rich domain may require p osttranslational modifications for activity. P1L, but not P2S, promoted RGD -mediated attachment of human gingival fibroblasts in a manner similar to t hat of native BSP, Deletion of the RGD domain or conversion of it to RGE (a rginine-glycine-glutamic acid) abolished the cell-attachment activity of P1 L. This suggests that, at least for human gingival fibroblasts, the major c ell-attachment activity in the recombinant BSP peptides studied (residues 4 2-87 and 69-300) requires the RGD sequence located at the C-terminal domain . (C) 2000 by Elsevier Science Inc. All rights reserved.