Adhesive and migratory effects of phosphophoryn are modulated by flanking peptides of the integrin binding motif.
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.
Department of Dental Science for Health Promotion, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan.
Shigeki
S
Suzuki
Department of Dental Science for Health Promotion, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan.
Seiji
S
Kobuke
Section of Orthodontics and Dentofacial Orthopedics, Faculty of Dental Science, Kyushu University, Fukuoka, Japan.
Naoto
N
Haruyama
Department of Dental Science for Health Promotion, Hiroshima University Institute of Biomedical and Health Sciences, Hiroshima, Japan.
Hiroaki
H
Hoshino
Functional Genomics Section, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America.
Ashok B
AB
Kulkarni
Section of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Japan.
Fusanori
F
Nishimura
D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D040461:Gene Components; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
C513308:Dmp1 protein, mouse; D016326:Extracellular Matrix Proteins; D016023:Integrins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C017630:phosphophoryn