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Adhesive and migratory effects of phosphophoryn are modulated by flanking peptides of the integrin binding motif.

Suzuki S, Kobuke S, Haruyama N, Hoshino H, Kulkarni AB, Nishimura F - PLoS ONE (2014)

Bottom Line: Furthermore, various point mutations in Ala482 and/or Ser483 converted recombinant PP into cell-adhesive proteins.Therefore, we concluded that the Ala482-Ser483 flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PP-RGD domain to be sequestered.The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin-mediated signaling molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Dental Science for Health Promotion, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan.

ABSTRACT
Phosphophoryn (PP) is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP). Gene duplications in the ancestor dentin matrix protein-1 (DMP-1) genomic sequence created the DSPP gene in toothed animals. PP and DMP-1 are phosphorylated extracellular matrix proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Many SIBLING members have been shown to evoke various cell responses through the integrin-binding Arg-Gly-Asp (RGD) domain; however, the RGD-dependent function of PP is not yet fully understood. We demonstrated that recombinant PP did not exhibit any obvious cell adhesion ability, whereas the simultaneously purified recombinant DMP-1 did. A cell adhesion inhibitory analysis was performed by pre-incubating human osteosarcoma MG63 cells with various PP peptides before seeding onto vitronectin. The results obtained revealed that the incorporation of more than one amino acid on both sides of the PP-RGD domain was unable to inhibit the adhesion of MG63 cells onto vitronectin. Furthermore, the inhibitory activity of a peptide containing the PP-RGD domain with an open carboxyl-terminal side (H-463SDESDTNSESANESGSRGDA482-OH) was more potent than that of a peptide containing the RGD domain with an open amino-terminal side (H-478SRGDASYTSDESSDDDNDSDSH499-OH). This phenomenon was supported by the potent cell adhesion and migration abilities of the recombinant truncated PP, which terminated with Ala482. Furthermore, various point mutations in Ala482 and/or Ser483 converted recombinant PP into cell-adhesive proteins. Therefore, we concluded that the Ala482-Ser483 flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PP-RGD domain to be sequestered. The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin-mediated signaling molecule.

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Six other mesenchymal cell lines were unable to attach to rPP.Three hDPC (human dental pulp cells) cells from different donors, Saos2 (human osteosarcoma cell line), parental MC3T3-E1 (heterogeneous cell population), and C2C12 (mouse myoblast cell line) were seeded onto rPP, rC-DMP-1, and vitronectin (100 nM) with MnCl2 (1 mM). The number of attached cells was evaluated as described before. Each value represents the mean of triplicate determinations; bars mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. ***p<0.001 indicates significantly lower and ++p<0.01 and +++p<0.001 indicate significantly higher than rC-DMP-1-coated wells.
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pone-0112490-g004: Six other mesenchymal cell lines were unable to attach to rPP.Three hDPC (human dental pulp cells) cells from different donors, Saos2 (human osteosarcoma cell line), parental MC3T3-E1 (heterogeneous cell population), and C2C12 (mouse myoblast cell line) were seeded onto rPP, rC-DMP-1, and vitronectin (100 nM) with MnCl2 (1 mM). The number of attached cells was evaluated as described before. Each value represents the mean of triplicate determinations; bars mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. ***p<0.001 indicates significantly lower and ++p<0.01 and +++p<0.001 indicate significantly higher than rC-DMP-1-coated wells.

Mentions: Since rPP was incapable of aiding cell adhesion to human osteosarcoma MG63 cells or mouse pre-osteoblastic MC3T3-E1 cells, we examined various cell adhesions on rPP in parallel with rC-DMP-1 and vitronectin with the addition of MnCl2. As shown in Figure 4, three human dental pulp (hDPC) cells, the human osteosarcoma cell line, SaoS2, parental heterogeneous MC3T3-E1 cells, and mouse myoblast cell line, C2C12 were clearly unable to attach to wells coated with 100 nM rPP, but did attach to wells coated with 100 nM rC-DMP-1 and vitronectin, which was similar to the results obtained for MG63 and MC3T3-E1 cells.


Adhesive and migratory effects of phosphophoryn are modulated by flanking peptides of the integrin binding motif.

Suzuki S, Kobuke S, Haruyama N, Hoshino H, Kulkarni AB, Nishimura F - PLoS ONE (2014)

Six other mesenchymal cell lines were unable to attach to rPP.Three hDPC (human dental pulp cells) cells from different donors, Saos2 (human osteosarcoma cell line), parental MC3T3-E1 (heterogeneous cell population), and C2C12 (mouse myoblast cell line) were seeded onto rPP, rC-DMP-1, and vitronectin (100 nM) with MnCl2 (1 mM). The number of attached cells was evaluated as described before. Each value represents the mean of triplicate determinations; bars mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. ***p<0.001 indicates significantly lower and ++p<0.01 and +++p<0.001 indicate significantly higher than rC-DMP-1-coated wells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4232355&req=5

pone-0112490-g004: Six other mesenchymal cell lines were unable to attach to rPP.Three hDPC (human dental pulp cells) cells from different donors, Saos2 (human osteosarcoma cell line), parental MC3T3-E1 (heterogeneous cell population), and C2C12 (mouse myoblast cell line) were seeded onto rPP, rC-DMP-1, and vitronectin (100 nM) with MnCl2 (1 mM). The number of attached cells was evaluated as described before. Each value represents the mean of triplicate determinations; bars mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. ***p<0.001 indicates significantly lower and ++p<0.01 and +++p<0.001 indicate significantly higher than rC-DMP-1-coated wells.
Mentions: Since rPP was incapable of aiding cell adhesion to human osteosarcoma MG63 cells or mouse pre-osteoblastic MC3T3-E1 cells, we examined various cell adhesions on rPP in parallel with rC-DMP-1 and vitronectin with the addition of MnCl2. As shown in Figure 4, three human dental pulp (hDPC) cells, the human osteosarcoma cell line, SaoS2, parental heterogeneous MC3T3-E1 cells, and mouse myoblast cell line, C2C12 were clearly unable to attach to wells coated with 100 nM rPP, but did attach to wells coated with 100 nM rC-DMP-1 and vitronectin, which was similar to the results obtained for MG63 and MC3T3-E1 cells.

Bottom Line: Furthermore, various point mutations in Ala482 and/or Ser483 converted recombinant PP into cell-adhesive proteins.Therefore, we concluded that the Ala482-Ser483 flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PP-RGD domain to be sequestered.The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin-mediated signaling molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Dental Science for Health Promotion, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan.

ABSTRACT
Phosphophoryn (PP) is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP). Gene duplications in the ancestor dentin matrix protein-1 (DMP-1) genomic sequence created the DSPP gene in toothed animals. PP and DMP-1 are phosphorylated extracellular matrix proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Many SIBLING members have been shown to evoke various cell responses through the integrin-binding Arg-Gly-Asp (RGD) domain; however, the RGD-dependent function of PP is not yet fully understood. We demonstrated that recombinant PP did not exhibit any obvious cell adhesion ability, whereas the simultaneously purified recombinant DMP-1 did. A cell adhesion inhibitory analysis was performed by pre-incubating human osteosarcoma MG63 cells with various PP peptides before seeding onto vitronectin. The results obtained revealed that the incorporation of more than one amino acid on both sides of the PP-RGD domain was unable to inhibit the adhesion of MG63 cells onto vitronectin. Furthermore, the inhibitory activity of a peptide containing the PP-RGD domain with an open carboxyl-terminal side (H-463SDESDTNSESANESGSRGDA482-OH) was more potent than that of a peptide containing the RGD domain with an open amino-terminal side (H-478SRGDASYTSDESSDDDNDSDSH499-OH). This phenomenon was supported by the potent cell adhesion and migration abilities of the recombinant truncated PP, which terminated with Ala482. Furthermore, various point mutations in Ala482 and/or Ser483 converted recombinant PP into cell-adhesive proteins. Therefore, we concluded that the Ala482-Ser483 flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PP-RGD domain to be sequestered. The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin-mediated signaling molecule.

Show MeSH
Related in: MedlinePlus