Ameloblastin (AMBN), an important component of the self-assembled enamel extra cellular matrix, contains several in silico predicted phosphorylation sites. However, to what extent these sites actually are phosphorylated and the possible effects of such post-translational modifications are still largely unknown. Here we report on in vitro experiments aimed at investigating what sites in AMBN are phosphorylated by casein kinase 2 (CK2) and protein kinase A (PKA) and the impact such phosphorylation has on self-assembly and calcium binding. All predicted sites in AMBN can be phosphorylated by CK2 and/or PKA. The experiments show that phosphorylation, especially in the exon 5 derived part of the molecule, is inversely correlated with AMBN self-assembly. These results support earlier findings suggesting that AMBN self-assembly is mostly dependent on the exon 5 encoded region of the AMBN gene. Phosphorylation was significantly more efficient when the AMBN molecules were in solution and not present as supramolecular assemblies, suggesting that post-translational modification of AMBN must take place before the enamel matrix molecules self-assemble inside the ameloblast cell. Moreover, phosphorylation of exon 5, and the consequent reduction in self-assembly, seem to reduce the calcium binding capacity of AMBN suggesting that post-translational modification of AMBN also can be involved in control of free Ca2+ during enamel extra cellular matrix biomineralization. Finally, it is speculated that phosphorylation can provide a functional crossroad for AMBN either to be phosphorylated and act as monomeric signal molecule during early odontogenesis and bone formation, or escape phosphorylation to be subsequently secreted as supramolecular assemblies that partake in enamel matrix structure and mineralization.

Department of Bioinformatics Institute of Organic Chemistry and Biochemistry Czech Academy of SciencesPrague Czechia

Department of Biomaterials Institute of Clinical Dentistry University of OsloOslo Norway

Division of Molecular Nutrition Department of Nutrition University of OsloOslo Norway

Section for Biochemistry and Molecular Biology Department of Biosciences University of OsloOslo Norway

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Bartlett J. D., Ganss B., Goldberg M., Moradian-Oldak J., Paine M. L., Snead M. L., et al. . (2006). 3. Protein-protein interactions of the developing enamel matrix. Curr. Top. Dev. Biol. 74, 57–115. 10.1016/S0070-2153(06)74003-0 PubMed DOI

Bawden J. W., Moran R. A., Deaton T. G., Saour C. M. (1996). Immunohistochemical localization of signal transduction pathways during amelogenesis: an initial exploration. Adv. Dent. Res. 10, 105–110. 10.1177/08959374960100020101 PubMed DOI

Bradford M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 10.1016/0003-2697(76)90527-3 PubMed DOI

Cerny R., Slaby I., Hammarstrom L., Wurtz T. (1996). A novel gene expressed in rat ameloblasts codes for proteins with cell binding domains. J. Bone Miner. Res. 11, 883–891. 10.1002/jbmr.5650110703 PubMed DOI

Chun Y. H., Lu Y., Hu Y., Krebsbach P. H., Yamada Y., Hu J. C., et al. . (2010a). Transgenic rescue of enamel phenotype in Ambn null mice. J. Dent. Res. 89, 1414–1420. 10.1177/0022034510379223 PubMed DOI PMC

Chun Y. H., Yamakoshi Y., Yamakoshi F., Fukae M., Hu J. C., Bartlett J. D., et al. . (2010b). Cleavage site specificity of MMP-20 for secretory-stage ameloblastin. J. Dent. Res. 89, 785–790. 10.1177/0022034510366903 PubMed DOI PMC

Contaxis C. C., Reithel F. J. (1971). Studies on Protein Multimers. II study of mechanism of urease dissociation in 1,2-propanediol - comparative studies with ethylene glycol and glycerol. J. Biol. Chem. 246, 677–685. PubMed

Delsuc F., Gasse B., Sire J.-Y. (2015). Evolutionary analysis of selective constraints identifies ameloblastin (AMBN) as a potential candidate for amelogenesis imperfecta. BMC Evol. Biol. 15:148. 10.1186/s12862-015-0431-0 PubMed DOI PMC

Fincham A. G., Moradianoldak J., Simmer J. P., Sarte P., Lau E. C., Diekwisch T., et al. . (1994). Self-assembly of a recombinant amelogenin protein generates supramolecular structures. J. Struct. Biol. 112, 103–109. 10.1006/jsbi.1994.1011 PubMed DOI

Fong C. D., Cerny R., Hammarstrom L., Slaby I. (1998). Sequential expression of an amelin gene in mesenchymal and epithelial cells during odontogenesis in rats. Eur. J. Oral Sci. 106(Suppl. 1), 324–330. 10.1111/j.1600-0722.1998.tb02193.x PubMed DOI

Fukae M., Kanazashi M., Nagano T., Tanabe T., Oida S., Gomi K. (2006). Porcine sheath proteins show periodontal ligament regeneration activity. Eur. J. Oral Sci. 114(Suppl 1), 212–218; discussion: 254–216, 381–212. 10.1111/j.1600-0722.2006.00309.x PubMed DOI

Fukumoto S., Kiba T., Hall B., Iehara N., Nakamura T., Longenecker G., et al. . (2004). Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts. J. Cell Biol. 167, 973–983. 10.1083/jcb.200409077 PubMed DOI PMC

Fukumoto S., Yamada A., Nonaka K., Yamada Y. (2005). Essential roles of ameloblastin in maintaining ameloblast differentiation and enamel formation. Cells Tissues Organs 181, 189–195. 10.1159/000091380 PubMed DOI

Geng S., White S. N., Paine M. L., Snead M. L. (2015). Protein interaction between ameloblastin and proteasome subunit alpha type 3 can facilitate redistribution of ameloblastin domains within forming enamel. J. Biol. Chem. 290, 20661–20673. 10.1074/jbc.M115.640185 PubMed DOI PMC

Iizuka S., Kudo Y., Yoshida M., Tsunematsu T., Yoshiko Y., Uchida T., et al. . (2011). Ameloblastin regulates osteogenic differentiation by inhibiting Src kinase via cross talk between integrin beta1 and CD63. Mol. Cell. Biol. 31, 783–792. 10.1128/MCB.00912-10 PubMed DOI PMC

Iwata T., Yamakoshi Y., Hu J. C., Ishikawa I., Bartlett J. D., Krebsbach P. H., et al. . (2007). Processing of ameloblastin by MMP-20. J. Dent. Res. 86, 153–157. 10.1177/154405910708600209 PubMed DOI

Kawasaki K., Buchanan A. V., Weiss K. M. (2007). Gene duplication and the evolution of vertebrate skeletal mineralization. Cells Tissues Organs 186, 7–24. 10.1159/000102678 PubMed DOI

Kitagawa M., Kitagawa S., Nagasaki A., Miyauchi M., Uchida T., Takata T. (2011). Synthetic ameloblastin peptide stimulates differentiation of human periodontal ligament cells. Arch. Oral Biol. 56, 374–379. 10.1016/j.archoralbio.2010.10.012 PubMed DOI

Lee S. K., Kim S. M., Lee Y. J., Yamada K. M., Yamada Y., Chi J. G. (2003). The structure of the rat ameloblastin gene and its expression in amelogenesis. Mol. Cells 15, 216–225. PubMed

Lu X., Fukumoto S., Yamada Y., Evans C. A., Diekwisch T. G., Luan X. (2016a). Ameloblastin, an extracellular matrix protein, affects long bone growth and mineralization. J. Bone Miner. Res. 31, 1235–1246. 10.1002/jbmr.2788 PubMed DOI

Lu X., Ito Y., Kulkarni A., Gibson C., Luan X., Diekwisch T. G. (2011). Ameloblastin-rich enamel matrix favors short and randomly oriented apatite crystals. Eur. J. Oral Sci. 119(Suppl. 1), 254–260. 10.1111/j.1600-0722.2011.00905.x PubMed DOI PMC

Lu X., Li W., Fukumoto S., Yamada Y., Evans C. A., Diekwisch T., et al. . (2016b). The ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing. Matrix Biol. 52–54, 113–126. 10.1016/j.matbio.2016.02.007 PubMed DOI PMC

Lyngstadaas S. P., Lundberg E., Ekdahl H., Andersson C., Gestrelius S. (2001). Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative. J. Clin. Periodontol. 28, 181–188. 10.1034/j.1600-051x.2001.028002181.x PubMed DOI

Ma P., Yan W., Tian Y., He J., Brookes S. J., Wang X. (2016). The importance of serine phosphorylation of ameloblastin on enamel formation. J. Dent. Res. 95, 1408–1414. 10.1177/0022034516661513 PubMed DOI PMC

Moradian-Oldak J., Bouropoulos N., Wang L., Gharakhanian N. (2002). Analysis of self-assembly and apatite binding properties of amelogenin proteins lacking the hydrophilic C-terminal. Matrix Biol. 21, 197–205. 10.1016/S0945-053X(01)00190-1 PubMed DOI

Murakami C., Dohi N., Fukae M., Tanabe T., Yamakoshi Y., Wakida K., et al. . (1997). Immunochemical and immunohistochemical study of the 27- and 29-kDa calcium-binding proteins and related proteins in the porcine tooth germ. Histochem. Cell Biol. 107, 485–494. 10.1007/s004180050136 PubMed DOI

Nakamura Y., Slaby I., Spahr A., Pezeshki G., Matsumoto K., Lyngstadaas S. P. (2006). Ameloblastin fusion protein enhances pulpal healing and dentin formation in porcine teeth. Calcif. Tissue Int. 78, 278–284. 10.1007/s00223-005-0144-2 PubMed DOI

Nanci A., Zalzal S., Lavoie P., Kunikata M., Chen W., Krebsbach P. H., et al. . (1998). Comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and amelogenin in rat incisors. J. Histochem. Cytochem. 46, 911–934. 10.1177/002215549804600806 PubMed DOI

Paine M. L., Snead M. L. (1997). Protein interactions during assembly of the enamel organic extracellular matrix. J. Bone Miner. Res. 12, 221–227. 10.1359/jbmr.1997.12.2.221 PubMed DOI

Paine M. L., Zhu D.-H., Luo W., Bringas P., Goldberg M., White S. N., et al. . (2000). Enamel biomineralization defects result from alterations to amelogenin self-assembly. J. Struct. Biol. 132, 191–200. 10.1006/jsbi.2000.4324 PubMed DOI

Perdigao P. F., Carvalho V. M., De Marco L., Gomez R. S. (2009). Mutation of ameloblastin gene in calcifying epithelial odontogenic tumor. Anticancer Res. 29, 3065–3067. PubMed

Perdigao P. F., Gomez R. S., Pimenta F. J., De Marco L. (2004). Ameloblastin gene (AMBN) mutations associated with epithelial odontogenic tumors. Oral Oncol. 40, 841–846. 10.1016/j.oraloncology.2004.03.004 PubMed DOI

Poulter J. A., Murillo G., Brookes S. J., Smith C. E., Parry D. A., Silva S., et al. . (2014). Deletion of ameloblastin exon 6 is associated with amelogenesis imperfecta. Hum. Mol. Genet. 23, 5317–5324. 10.1093/hmg/ddu247 PubMed DOI PMC

Putnam C. D., Hammel M., Hura G. L., Tainer J. A. (2007). X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q. Rev. Biophys. 40, 191–285. 10.1017/S0033583507004635 PubMed DOI

Robinson C., Brookes S. J., Shore R. C., Kirkham J. (1998). The developing enamel matrix: nature and function. Eur. J. Oral Sci. 106(Suppl. 1), 282–291. 10.1111/j.1600-0722.1998.tb02188.x PubMed DOI

Sonoda A., Iwamoto T., Nakamura T., Fukumoto E., Yoshizaki K., Yamada A., et al. . (2009). Critical role of heparin binding domains of ameloblastin for dental epithelium cell adhesion and ameloblastoma proliferation. J. Biol. Chem. 284, 27176–27184. 10.1074/jbc.M109.033464 PubMed DOI PMC

Spahr A., Lyngstadaas S. P., Slaby I., Pezeshki G. (2006). Ameloblastin expression during craniofacial bone formation in rats. Eur. J. Oral Sci. 114, 504–511. 10.1111/j.1600-0722.2006.00403.x PubMed DOI

Stakkestad Ø., Lyngstadaas S. P., Vondrasek J., Gordeladze J. O., Reseland J. E. (2017). Ameloblastin peptides modulates the osteogenic capacity of human mesenchymal stem cells. Front. Physiol. 8:58. 10.3389/fphys.2017.00058 PubMed DOI PMC

Su J., Chandrababu K. B., Moradian-Oldak J. (2016). Ameloblastin peptide encoded by exon 5 interacts with amelogenin N-terminus. Biochem. Biophys. Rep. 7, 26–32. 10.1016/j.bbrep.2016.05.007 PubMed DOI PMC

Takahashi K., Shimonishi M., Wang R., Watanabe H., Kikuchi M. (2012). Epithelial-mesenchymal interactions induce enamel matrix proteins and proteases in the epithelial cells of the rests of Malassez in vitro. Eur. J. Oral Sci. 120, 475–483. 10.1111/j.1600-0722.2012.01002.x PubMed DOI

Tamburstuen M. V., Reppe S., Spahr A., Sabetrasekh R., Kvalheim G., Slaby I., et al. . (2010). Ameloblastin promotes bone growth by enhancing proliferation of progenitor cells and by stimulating immunoregulators. Eur. J. Oral Sci. 118, 451–459. 10.1111/j.1600-0722.2010.00760.x PubMed DOI

Tamburstuen M. V., Reseland J. E., Spahr A., Brookes S. J., Kvalheim G., Slaby I., et al. . (2011a). Ameloblastin expression and putative autoregulation in mesenchymal cells suggest a role in early bone formation and repair. Bone 48, 406–413. 10.1016/j.bone.2010.09.007 PubMed DOI PMC

Tamburstuen M. V., Snead M. L., Reseland J. E., Paine M. L., Lyngstadaas S. P. (2011b). Ameloblastin upstream region contains structural elements regulating transcriptional activity in a stromal cell line derived from bone marrow. Eur. J. Oral Sci. 119(Suppl. 1), 286–292. 10.1111/j.1600-0722.2011.00910.x PubMed DOI PMC

Toyosawa S., Fujiwara T., Ooshima T., Shintani S., Sato A., Ogawa Y., et al. . (2000). Cloning and characterization of the human ameloblastin gene. Gene 256, 1–11. 10.1016/s0378-1119(00)00379-6 PubMed DOI

Uchida T., Murakami C., Dohi N., Wakida K., Satoda T., Takahashi O. (1997). Synthesis, secretion, degradation, and fate of ameloblastin during the matrix formation stage of the rat incisor as shown by immunocytochemistry and immunochemistry using region-specific antibodies. J. Histochem. Cytochem. 45, 1329–1340. 10.1177/002215549704501002 PubMed DOI

Uchida T., Murakami C., Wakida K., Dohi N., Iwai Y., Simmer J. P., et al. . (1998). Sheath proteins: synthesis, secretion, degradation and fate in forming enamel. Eur. J. Oral Sci. 106(Suppl. 1), 308–314. 10.1111/j.1600-0722.1998.tb02191.x PubMed DOI

Vymetal J., Slaby I., Spahr A., Vondrasek J., Lyngstadaas S. P. (2008). Bioinformatic analysis and molecular modelling of human ameloblastin suggest a two-domain intrinsically unstructured calcium-binding protein. Eur. J. Oral Sci. 116, 124–134. 10.1111/j.1600-0722.2008.00526.x PubMed DOI

Wald T., Bednarova L., Osicka R., Pachl P., Sulc M., Lyngstadaas S. P., et al. . (2011). Biophysical characterization of recombinant human ameloblastin. Eur. J. Oral Sci. 119(Suppl. 1), 261–269. 10.1111/j.1600-0722.2011.00913.x PubMed DOI

Wald T., Osickova A., Sulc M., Benada O., Semeradtova A., Rezabkova L., et al. . (2013). Intrinsically disordered enamel matrix protein ameloblastin forms ribbon-like supramolecular structures via an N-terminal segment encoded by exon 5. J. Biol. Chem. 288, 22333–22345. 10.1074/jbc.M113.456012 PubMed DOI PMC

Wald T., Spoutil F., Osickova A., Prochazkova M., Benada O., Kasparek P., et al. . (2017). Intrinsically disordered proteins drive enamel formation via an evolutionarily conserved self-assembly motif. Proc. Natl. Acad. Sci. U.S.A. 114, E1641–E1650. 10.1073/pnas.1615334114 PubMed DOI PMC

Wazen R. M., Moffatt P., Zalzal S. F., Yamada Y., Nanci A. (2009). A mouse model expressing a truncated form of ameloblastin exhibits dental and junctional epithelium defects. Matrix Biol. 28, 292–303. 10.1016/j.matbio.2009.04.004 PubMed DOI PMC

Yamakoshi Y., Tanabe T., Oida S., Hu C. C., Simmer J. P., Fukae M. (2001). Calcium binding of enamel proteins and their derivatives with emphasis on the calcium-binding domain of porcine sheathlin. Arch. Oral Biol. 46, 1005–1014. 10.1016/S0003-9969(01)00070-X PubMed DOI

Zhang X., Diekwisch T. G. H., Luan X. H. (2011). Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse. Eur. J. Oral Sci. 119, 270–279. 10.1111/j.1600-0722.2011.00889.x PubMed DOI PMC

Proteolytic profiles of two isoforms of human AMBN expressed in E. coli by MMP-20 and KLK-4 proteases

Early evolution of enamel matrix proteins is reflected by pleiotropy of physiological functions

Characterization of AMBN I and II Isoforms and Study of Their Ca2+-Binding Properties