Structure-activity relationship of human bone sialoprotein peptides

Abstract

In the current study, the relationship between the structure of the RGD-containing human bone sialoprotein (hBSP) peptide 278-293 and its attachment activity toward osteoblast-like (MC3T3) cells was investigated. This goal was accomplished by examining the comparative cell-attachment activities of several truncated forms of peptide 278-293. Computer modeling of the various peptides was also performed to assess the role of secondary structure in peptide bioactivity. Elimination of tyrosine-278 at the N-terminus resulted in a more dramatic loss of cell-attachment activity compared with the removal of either tyrosine-293 or the arg-ala-tyr (291-293) tripeptide. Although replacement of the RGD (arg-gly-asp) peptide moiety with peptide KAE (lys-ala-glu) resulted in a dramatic loss of cell-attachment activity, a peptide containing RGE (arg-gly-glu) in place of RGD retained 70-85% of the parental peptide's attachment activity. These results suggest that the N-terminal RGD-flanking region of hBSP peptide 278-293, in particular the tyrosine-278 residue, represents a second cell-attachment site that stabilizes the RGD-integrin receptor complex. Computer modeling also suggested that a β-turn encompassing RGD or RGE in some of the hBSP peptides may facilitate its binding to integrins by increasing the exposure of the tripeptide. This knowledge may be useful in the future design of biomimetic peptides which are more effective in promoting the attachment of osteogenic cells to implant surfaces in vivo.

Keywords: RGD peptide; biomimetic; extracellular matrix; integrins; osteoblasts.

FIGURE 1

Sequences of human BSP peptides (hBSP). The comparative sequences of BSP residues 278-293 (P3), 281-290 (P2), 281-293 (DB1), 278-290 (DB2), 279-293 (DB3), 278-292 (DB4) and 279-292 (DB5) are shown with the RGD core sequence (286-288) as a reference point. The comparative sequences of two variants of hBSP peptide 278-292 modified within the RGD sequence, including the replacement of aspartic acid with glutamic acid at residue 288 (DB4-E) and the replacement of arg-gly-asp at 286-288 with lys-ala-glu (DB4-KAE) ; and a third peptide modified by the sulfation of tyrosines 278 and 290 (DB4-sulfate), are also shown.

FIGURE 2

Relative potencies of hBSP peptides P3 and P2, DB1, DB2, DB3, DB4, DB5 for stimulating the attachment of MC3T3 cells. (A) The number of attached cells measured in the wells of nontissue culture plates coated with either peptide as described in Methods is expressed as a percentage of the number of attached cells measured in control wells that were not coated with peptide (control number of attached cells = 2411 ± 13 cells per well; mean ± SEM; N = 9). Data presented as mean ± SEM for control and for each coating concentration of peptide were obtained from at least three independent cell cultures (N = at least 3). *P < 0.001 compared to control and P < 0.04 compared to DB1, DB3, DB5 or P2 at every peptide coating concentration shown based on student's t test. **P < 0.001 or less compared to control. ***P < 0.007 or less compared to control for every concentration tested. ****P < 0.02 or less compared to control for every concentration tested. *****P < 0.02 or less compared to control for peptide coating concentrations (shown on X axis) 25 – 200 uM. (B) For each hBSP peptide, the number of attached cells was also plotted vs. the adsorbed peptide's surface concentration measured by protein assay.

FIGURE 3

Relative potencies of hBSP peptides P3 and P2 for stimulating the attachment of MC3T3 and MG63 cells. (A) The number of attached cells measured in the wells of nontissue culture plates coated with either peptide as described in Methods is expressed as a percentage of the number of attached cells measured in control wells that were not coated with peptide. The data for attachment of MC3T3 cells to peptides was taken from Fig. 2. For MG63 cells, control number of attached cells = 3624 ± 390 cells per well; mean ± SEM; N = 6. Data presented as mean ± SEM for each peptide coating concentration were obtained from at least three independent cell cultures (N = at least 3). *P < 0.001 or less compared to control and P < 0.04 compared to P2 at every peptide coating concentration shown based on student's t test. **P < 0.001 or less compared to control and P < 0.05 compared to P2 at every peptide coating concentration shown based on student's t test. (B) For each hBSP peptide, the number of attached cells was also plotted vs. the adsorbed peptide's surface concentration measured by protein assay.

Figure 4

Inhibitory effects of soluble RGD tripeptide or anti-integrin antibodies on MC3T3 cell attachment to hBSP peptides. The effects of increasing concentrations of soluble (A) RGD or (B) anti-integrin (α_v or α₅β₁) antibodies, respectively, on the number of MC3T3 cells that attached to (A) P3, DB1 and DB2 (25 μM coating concentration) or (B) peptide P3 (10 μM) were measured. Cells were preincubated with a solution containing 0.1 – 500 μM of the tripeptide RGD or 1:500 to 1:25 dilutions of an anti-α_v or anti-α₅β₁ integrin antibody for 30 min. prior to addition of cells to the peptide-coated nontissue culture plates. The number of cells that attached to peptide-coated plates measured at each [RGD] or antibody dilution is expressed as a percentage of the control attached cell number (CONTROL) measured when plates were coated with peptides but cells were not preincubated with RGD (0 MICROMOLAR [RGD]) or antibodies (0 ANTIBODY DILUTION). Data are presented as mean ± SEM peptide (N = at least 3). *P < 0.01 or less for RGD concentrations of 100 and 500 μM compared to control (0 μM RGD) - DB1-coated plates. **P < 0.035 or less for RGD concentrations of 100 and 500 μM compared to control (0 μM RGD) - DB2-coated plates. ***P < 0.05 or less for RGD concentrations of 10, 100 and 500 μM compared to control (0 μM RGD) - P3-coated plates. ^a,bP < 0.007 or 0.0001, respectively, or less for every dilution of anti-α_v or anti-α₅β₁ integrin antibodies compared to control (0 ANTIBODY DILUTION) - P3-coated plates (based on student's t test).

FIGURE 5

Relative cell attachment potencies of DB4, DB4-E and DB4-KAE. (A) The number of attached cells measured in the wells of nontissue culture plates coated with either peptide is expressed as described under Figure 2 (control number of attached cells = 2803 ± 9 cells per well; mean ± SEM; N = 15). Data presented as mean ± SEM for control and for each coating concentration of peptide were obtained from at least three independent cell cultures (N = at least 3). *P < 0.001 compared to control at every peptide coating concentration shown based on student's t test. **P < 0.006 compared to control at every concentration shown and P < 0.05 compared to DB4-KAE-coated wells at 25 - 200 uM. ***P < 0.003 compared to control at every peptide coating concentration (shown on X axis). (B) For each hBSP peptide, the number of attached cells was also plotted vs. the adsorbed peptide's surface concentration measured by protein assay.

FIGURE 6

Per cent adsorption of DB4, DB4-E and DB4-KAE to 96-well plates. 96-well nontissue culture plates were coated with peptides and washed as described in Methods. The mean amount of adsorbed peptides measured at coating solution concentrations of 1, 10, 25, 50 and 100 ±M is expressed as % of solution phase peptide in moles (peptides added in solution phase of 200 ul/well) adsorbed to each well. If 100 % of the peptide molecules in a 100 ±M peptide solution adsorbed to the well in a 96-well culture plate, this would correspond to a peptide surface concentration of 70.8 nmoles / cm². Data presented as mean ± SEM for control and for each concentration (N = 3-4). *P < 0.04 for 10-50 Fig. μM DB4 compared to DB4-E and DB4-KAE based on student's t test. **P < 0.05 for 100 μM DB4 compared to DB4-E. and DB4-KAE.

FIGURE 7

Computer model-generated predictions of hBSP secondary structure. Secondary structures predicted with the aid of the I-Tasser server are shown for peptides P3, P2 and DB4-KAE. Regions of individual peptides predicted to contain an alpha helix secondary structure are shown in red. For peptide models containing areas of alpha helix conformation (P3 and DB3), the relative positions of polar and nonpolar residues outside of the alpha helix structure are shown in blue and grey, respectively. The N-terminus is located on the left-hand side of each of the four models shown.