Limits...
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.

Show MeSH

Related in: MedlinePlus

Replacement of the 1st and/or 2nd carboxyl-terminal amino acids of the PP-RGD domain altered its ability.Ninety-six-well plates were precoated with 250 nM or 1 µM of normal rPP-ΔSSD and various mutated rPP-ΔSSD proteins, seeded with MG63 cells in serum-free medium, and incubated for 1 hr. The number of attached cells was evaluated as described above. 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<0.05, **p<0.01, and ***p<0.001 indicate significantly higher than rPP-ΔSSD-coated wells at the same concentration.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4232355&req=5

pone-0112490-g009: Replacement of the 1st and/or 2nd carboxyl-terminal amino acids of the PP-RGD domain altered its ability.Ninety-six-well plates were precoated with 250 nM or 1 µM of normal rPP-ΔSSD and various mutated rPP-ΔSSD proteins, seeded with MG63 cells in serum-free medium, and incubated for 1 hr. The number of attached cells was evaluated as described above. 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<0.05, **p<0.01, and ***p<0.001 indicate significantly higher than rPP-ΔSSD-coated wells at the same concentration.

Mentions: Since the release of Ser483 from the vicinity of RGD disclosed PP-RGD ability, we speculated that the Ala-Ser peptide bond may be the key to sequestering the ability of the RGD domain to act on integrin. Therefore, we newly generated rPP-ΔSSD-RGDTSYT in which Ala482 was replaced with Thr, rPP-ΔSSD-RGDDSYT in which Ala482 was replaced with Asp, rPP-ΔSSD-RGDACYT in which Ser483 was replaced with Cys, rPP-ΔSSD-RGDAIYT in which Ser483 was replaced with Ile, rPP-ΔSSD-RGDAVYT in which Ser483 was replaced with Val, and rPP-ΔSSD-RGDNPYT in which Ala482-Ser483bond was replaced with an Asn-Pro bond to mimic the human and mouse DMP-1-RGD domains. We then examined their cell-adhesive potencies by quantifying the number of attached MG63 cells on wells coated with normal rPP-ΔSSD and these mutated rPP-ΔSSD proteins. As shown in Figure 9, MG63 cells were able to attach to the plates coated with 1 µM of these mutated rPP-ΔSSD proteins, with rPP-ΔSSD-RGDDSYT and rPP-ΔSSD-RGDAVYT showing higher binding capacities. In contrast, MG63 cells were unable to attach to plates coated with 1 µM of normal rPP-ΔSSD. The binding ability of MG63 cells was higher on plates coated with mutated rPP-ΔSSD proteins than with rPP-ΔSSD at 250 nM; however, the cell-adhesive effects of mutated rPP-ΔSSD proteins were not significant at this concentration.


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)

Replacement of the 1st and/or 2nd carboxyl-terminal amino acids of the PP-RGD domain altered its ability.Ninety-six-well plates were precoated with 250 nM or 1 µM of normal rPP-ΔSSD and various mutated rPP-ΔSSD proteins, seeded with MG63 cells in serum-free medium, and incubated for 1 hr. The number of attached cells was evaluated as described above. 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<0.05, **p<0.01, and ***p<0.001 indicate significantly higher than rPP-ΔSSD-coated wells at the same concentration.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112490-g009: Replacement of the 1st and/or 2nd carboxyl-terminal amino acids of the PP-RGD domain altered its ability.Ninety-six-well plates were precoated with 250 nM or 1 µM of normal rPP-ΔSSD and various mutated rPP-ΔSSD proteins, seeded with MG63 cells in serum-free medium, and incubated for 1 hr. The number of attached cells was evaluated as described above. 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<0.05, **p<0.01, and ***p<0.001 indicate significantly higher than rPP-ΔSSD-coated wells at the same concentration.
Mentions: Since the release of Ser483 from the vicinity of RGD disclosed PP-RGD ability, we speculated that the Ala-Ser peptide bond may be the key to sequestering the ability of the RGD domain to act on integrin. Therefore, we newly generated rPP-ΔSSD-RGDTSYT in which Ala482 was replaced with Thr, rPP-ΔSSD-RGDDSYT in which Ala482 was replaced with Asp, rPP-ΔSSD-RGDACYT in which Ser483 was replaced with Cys, rPP-ΔSSD-RGDAIYT in which Ser483 was replaced with Ile, rPP-ΔSSD-RGDAVYT in which Ser483 was replaced with Val, and rPP-ΔSSD-RGDNPYT in which Ala482-Ser483bond was replaced with an Asn-Pro bond to mimic the human and mouse DMP-1-RGD domains. We then examined their cell-adhesive potencies by quantifying the number of attached MG63 cells on wells coated with normal rPP-ΔSSD and these mutated rPP-ΔSSD proteins. As shown in Figure 9, MG63 cells were able to attach to the plates coated with 1 µM of these mutated rPP-ΔSSD proteins, with rPP-ΔSSD-RGDDSYT and rPP-ΔSSD-RGDAVYT showing higher binding capacities. In contrast, MG63 cells were unable to attach to plates coated with 1 µM of normal rPP-ΔSSD. The binding ability of MG63 cells was higher on plates coated with mutated rPP-ΔSSD proteins than with rPP-ΔSSD at 250 nM; however, the cell-adhesive effects of mutated rPP-ΔSSD proteins were not significant at this concentration.

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