Identification of Novel Amelogenin-Binding Proteins by Proteomics Analysis

Abstract

Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology. It is used in periodontal surgery to regenerate cementum, periodontal ligament, and alveolar bone. However, the precise molecular mechanisms underlying periodontal regeneration are still unclear. In this study, we investigated the proteins bound to amelogenin, which are suggested to play a pivotal role in promoting periodontal tissue regeneration. To identify new molecules that interact with amelogenin and are involved in osteoblast activation, we employed coupling affinity chromatography with proteomic analysis in fractionated SaOS-2 osteoblastic cell lysate. In SaOS-2 cells, many of the amelogenin-interacting proteins in the cytoplasm were mainly cytoskeletal proteins and several chaperone molecules of heat shock protein 70 (HSP70) family. On the other hand, the proteomic profiles of amelogenin-interacting proteins in the membrane fraction of the cell extracts were quite different from those of the cytosolic-fraction. They were mainly endoplasmic reticulum (ER)-associated proteins, with lesser quantities of mitochondrial proteins and nucleoprotein. Among the identified amelogenin-interacting proteins, we validated the biological interaction of amelogenin with glucose-regulated protein 78 (Grp78/Bip), which was identified in both cytosolic and membrane-enriched fractions. Confocal co-localization experiment strongly suggested that Grp78/Bip could be an amelogenin receptor candidate. Further biological evaluations were examined by Grp78/Bip knockdown analysis with and without amelogenin. Within the limits of the present study, the interaction of amelogenin with Grp78/Bip contributed to cell proliferation, rather than correlate with the osteogenic differentiation in SaOS-2 cells. Although the biological significance of other interactions are not yet explored, these findings suggest that the differential effects of amelogenin-derived osteoblast activation could be of potential clinical significance for understanding the cellular and molecular bases of amelogenin-induced periodontal tissue regeneration.

Grp78 Is Critical for Amelogenin‐Induced Cell Migration in a Multipotent Clonal Human Periodontal Ligament Cell Line

“…Cloning and expression of a glutathione S-transferase (GST) full-length recombinant amelogenin fusion (GST-rM180), and the purification of rM180 were previously described (Fukuda et al, 2013). Cloning and expression of a glutathione S-transferase (GST) full-length recombinant amelogenin fusion (GST-rM180), and the purification of rM180 were previously described (Fukuda et al, 2013).…”

“…Although the involvement of other enamel matrix proteins in the regeneration process cannot be excluded (Amin et al, 2012), recent studies have demonstrated that recombinant amelogenin induced the in vivo regeneration of all toothsupporting tissues (Haze et al, 2009). Using this proteomic screen we identified glucose-regulated protein 78 (Grp78) as an amelogenin-binding protein, present in both the cytosolic and membrane-enriched fractions of osteoblastic cells (Fukuda et al, 2013). Using this proteomic screen we identified glucose-regulated protein 78 (Grp78) as an amelogenin-binding protein, present in both the cytosolic and membrane-enriched fractions of osteoblastic cells (Fukuda et al, 2013).…”

New Therapeutic Strategy for Regenerating Periodontal Tissue Based on the Combination of Amelogenin and Reapplications of Existing Grp78 Inducer

“…Therefore, identification of amelogenin-binding molecules in cells, such as osteoblasts and mesenchymal stem cell (MSC)-derived PDL stem cells (PDLSCs), which are important in the regeneration of periodontal tissue, was an indispensable topic of study. By conducting a proteome analysis focused on amelogenin, the applicants were able to succeed, for the first time worldwide, in identifying a new amelogeninbinding proteins in osteoblasts that includes Grp78 [12]. Furthermore, we also found that the interaction between amelogenin and Grp78 not only enhances cell proliferation in osteoblasts [12] but also contribute to cell migration in PDLSCs [13].…”

“…By conducting a proteome analysis focused on amelogenin, the applicants were able to succeed, for the first time worldwide, in identifying a new amelogeninbinding proteins in osteoblasts that includes Grp78 [12]. Furthermore, we also found that the interaction between amelogenin and Grp78 not only enhances cell proliferation in osteoblasts [12] but also contribute to cell migration in PDLSCs [13].…”

“…Indeed, we have found that Grp78 participates in the promotion of osteoblast proliferation [12] and periodontal ligament stem cell migration [13] via an amelogenin stimulus. Grp78 is reported to have two anti-inflammatory pathways.…”