Ameloblastin (AMBN) cleavage products are the most abundant non-amelogenin proteins in the enamel matrix of developing teeth. AMBN N-terminal cleavage products accumulate in the sheath space between enamel rods, while AMBN C-terminal products localize within rods. We tested the hypothesis that MMP-20 is the protease that cleaves AMBN. Glycosylated recombinant porcine AMBN (rpAMBN) was expressed in human kidney 293F cells, and recombinant porcine enamelysin (rpMMP-20) was expressed in bacteria. The purified proteins were incubated together at an enzyme:substrate ratio of 1:100. N-terminal sequencing of AMBN digestion products determined that rpMMP-20 cleaved rpAMBN after Pro2, Gln130, Gln139, Arg170, and Ala222. This shows that MMP-20 generates the 23-kDa AMBN starting at Tyr223, as well as the 17-kDa (Val1-Arg170) and 15-kDa (Val1-Gln130) AMBN cleavage products that concentrate in the sheath space during the secretory stage. We conclude that MMP-20 processes ameloblastin in vitro and in vivo.
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References
1.
Akita H, Fukae M, Shimoda S, Aoba T -1992-. Localization of glycosylated matrix proteins in secretory porcine enamel and their possible functional roles in enamel mineralization. Arch Oral Biol37:953–962.
Bartlett JD, Ryu OH, Xue J, Simmer JP, Margolis HC -1998-. Enamelysin mRNA displays a developmentally defined pattern of expression and encodes a protein which degrades amelogenin. Connect Tissue Res39:101–109.
5.
Brookes SJ, Kirkham J, Shore RC, Wood SR, Slaby I, Robinson C -2001-. Amelin extracellular processing and aggregation during rat incisor amelogenesis. Arch Oral Biol46:201–208.
6.
Caterina JJ, Skobe Z, Shi J, Ding Y, Simmer JP, Birkedal-Hansen H, et al. -2002-. Enamelysin -matrix metalloproteinase 20--deficient mice display an amelogenesis imperfecta phenotype. J Biol Chem277:49598–49604.
7.
Fincham AG, Moradian-Oldak J, Sarte PE -1994-. Mass-spectrographic analysis of a porcine amelogenin identifies a single phosphorylated locus. Calcif Tissue Int55:398–400.
8.
Fincham AG, Moradian-Oldak J, Simmer JP -1999-. The structural biology of the developing dental enamel matrix. J Struct Biol126:270–299.
9.
Fukae M, Tanabe T -1987a-. Nonamelogenin components of porcine enamel in the protein fraction free from the enamel crystals. Calcif Tissue Int40:286–293.
10.
Fukae M, Tanabe T -1987b-. 45Ca-labeled proteins found in porcine developing dental enamel at an early stage of development. Adv Dent Res1:261–266.
11.
Fukae M, Kanazashi M, Nagano T, Tanabe T, Oida S, Gomi K -2006-. Porcine sheath proteins show periodontal ligament regeneration activity. Eur J Oral Sci114-Suppl 1-:212–218; discussion 254–216, 381–312.
12.
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 Biol167:973–983.
13.
Hart PS, Hart TC, Michalec MD, Ryu OH, Simmons D, Hong S, et al. -2004-. Mutation in kallikrein 4 causes autosomal recessive hypomaturation amelogenesis imperfecta. J Med Genet41:545–549.
14.
Hu CC, Fukae M, Uchida T, Qian Q, Zhang CH, Ryu OH, et al. -1997-. Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins. J Dent Res76:648–657.
15.
Hu JC, Sun X, Zhang C, Liu S, Bartlett JD, Simmer JP -2002-. Enamelysin and kallikrein-4 mRNA expression in developing mouse molars. Eur J Oral Sci110:307–315.
16.
Hu JC, Yamakoshi Y, Yamakoshi F, Krebsbach PH, Simmer JP -2005-. Proteomics and genetics of dental enamel. Cells Tissues Organs181:219–231.
17.
Kim JW, Simmer JP, Hart TC, Hart PS, Ramaswami MD, Bartlett JD, et al. -2005-. MMP-20 mutation in autosomal recessive pigmented hypomaturation amelogenesis imperfecta. J Med Genet42:271–275.
18.
Kim JW, Simmer JP, Lin BP, Seymen F, Bartlett JD, Hu JC -2006-. Mutational analysis of candidate genes in 24 amelogenesis imperfecta families. Eur J Oral Sci114-Suppl 1-:3–12.
19.
Lyngstadaas SP -2001-. Synthetic hammerhead ribozymes as tools in gene expression. Crit Rev Oral Biol Med12:469–478.
20.
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 Biol107:485–494.
21.
Ozdemir D, Hart PS, Ryu OH, Choi SJ, Ozdemir-Karatas M, Firatli E, et al. -2005-. MMP20 active-site mutation in hypomaturation amelogenesis imperfecta. J Dent Res84:1031–1035.
22.
Ryu OH, Fincham AG, Hu CC, Zhang C, Qian Q, Bartlett JD, et al. -1999-. Characterization of recombinant pig enamelysin activity and cleavage of recombinant pig and mouse amelogenins. J Dent Res78:743–750.
23.
Ryu O, Hu JC, Yamakoshi Y, Villemain JL, Cao X, Zhang C, et al. -2002-. Porcine kallikrein-4 activation, glycosylation, activity, and expression in prokaryotic and eukaryotic hosts. Eur J Oral Sci110:358–365.
24.
Shimizu M -1984-. Investigation of highly aggregative components in porcine enamel proteins. In: Tooth enamel IV. Fearnhead RW, Suga S, editors. New York: Elsevier Science Publishers, pp. 156–160.
25.
Simmer J -2004-. Prostase. In: Handbook of proteolytic enzymes. Barrett A, Rawlings N, Woessner J, editors. Amsterdam: Academic Press, pp. 1612–1613.
26.
Simmer JP, Hu JC -2002-. Expression, structure, and function of enamel proteinases. Connect Tissue Res43:441–449.
27.
Simmer JP, Sun X, Yamada Y, Zhang CH, Bartlett JD, Hu JC-C -2004-. Enamelysin and kallikrein-4 expression in the mouse incisor. In: Biomineralization: formation, diversity, evolution and application. Proceedings of the 8th International Symposium on Biomineralization, Niigata, Japan, Sept 25–28, 2001. Kobayashi I, Ozawa H, editors. Hadano, Japan: Tokai University Press, pp. 348–352.
28.
Uchida T, Tanabe T, Fukae M, Shimizu M, Yamada M, Miake K, et al. -1991-. Immunochemical and immunohistochemical studies, using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13–17 kDa nonamelogenins, on immature enamel of the pig and rat. Histochemistry96:129–138.
29.
Uchida T, Fukae M, Tanabe T, Yamakoshi Y, Satoda T, Murakami C, et al. -1995-. Immunochemical and immunocytochemical study of a 15 kDa non-amelogenin and related proteins in the porcine immature enamel: proposal of a new group of enamel proteins sheath proteins. Biomed Res16:131–140.
30.
Uchida T, Murakami C, Wakida K, Dohi N, Iwai Y, Simmer JP, et al. -1998-. Sheath proteins: synthesis, secretion, degradation and fate in forming enamel. Eur J Oral Sci106-Suppl 1-:308–314.
31.
Yamakoshi Y, Tanabe T, Oida S, Hu CC, Simmer JP, Fukae M -2001-. Calcium binding of enamel proteins and their derivatives with emphasis on the calcium-binding domain of porcine sheathlin. Arch Oral Biol46:1005–1014.
32.
Yamakoshi Y, Hu JC-C, Ryu OH, Tanabe T, Oida S, Fukae M, et al. -2004-. A comprehensive strategy for purifying pig enamel proteins. In: Biomineralization: formation, diversity, evolution and application. Proceedings of the 8th International Symposium on Biomineralization, Niigata, Japan, Sept 25–28, 2001. Kobayashi I, Ozawa H, editors. Hadano, Japan: Tokai University Press, pp. 326–332.
33.
Yamakoshi Y, Zhang H, Hu JC-C, Iwata T, Simmer JP -2006-. Proteomic analysis of enamel matrix using a two-dimensional protein fractionation system. Eur J Oral Sci114-Suppl 1-:266–271.
34.
Zeichner-David M, Chen LS, Hsu Z, Reyna J, Caton J, Bringas P -2006-. Amelogenin and ameloblastin show growth-factor like activity in periodontal ligament cells. Eur J Oral Sci114-Suppl 1-:244–253; discussion 254–246, 381–342.
