Structure-activity relationship of osteoblast adhesive human Bone Sialoprotein peptides

RGD-biomimetic peptides can be used to promote successful biointegration of prosthetic implant materials. Previously, we reported on enhanced osteoblast-like (MC3T3-E1) cell attachment with several RGD-containing peptides of human bone sialoprotein (hBSP), specifically to tyrosine-rich hBSP peptides 278-302 and 278-293. Purpose: To examine further modifications of hBSP peptides and identify core domain(s) essential for cell adhesive activity by examining the comparative adhesive effects of several truncated forms of peptide 278-293. Methods: hBSP peptides were produced with FMOC chemistry and tested with osteoblast MC3T3 cells quantitated on non-tissue culture polystyrene by assay of hexosaminidase. Results: hBSP peptides 278-290 (DB2) and 278-292 (DB4) demonstrated the greatest maximal stimulation (600-650 % of control) of cell attachment. The key distinguishing structural characteristic shared among these more adhesive peptides was the presence of tyrosine-278 at the N-terminus. Tyrosine sulfation of peptide 278-292 did not affect cell adhesive potency. A modified hBSP peptide 278-292, in which an aspartic acid residue at position 288 (D of the RGD sequence) was replaced by a glutamic acid residue (to form RGE), showed no loss of adhesive activity. In contrast, the replacement of the RGD with KAE resulted in the loss of over 50 % of adhesive effectiveness. Finally, the addition of an 8-residue polyglutamic acid sequence at either the N or C-terminus of peptide 278-292 had no effect on the molecule’s cell adhesive properties. Conclusion: This study has shown that an intact RGD region and the N-terminal RGD-flanking region of hBSP peptide 278-293, in particular the tyrosine-278 residue, are important for the molecule’s cell adhesive activity. The C-terminal RGD-flanking region, tyrosine sulfation or peptide length are not important. This knowledge will be useful in the future design of biomimetic peptides to promote osseointegration of prosthetic implants in vivo. Supported by VA Merit Grant 2894-005