Reduced Amelogenin-MMP20 Interactions in Amelogenesis Imperfecta

Tanimoto, Kotaro; Le, Thuan; Zhu, Liu; Witkowska, H. Ewa; Robinson, S.; Hall, S.; Hwang, Peter K.; DenBesten, Pamela; Li, W.

doi:10.1177/154405910808700516

Cited by 16 publications

(16 citation statements)

References 30 publications

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“…The T21I mutation destabilizes the disordered structure, and in turn, the relative population of partially folded soluble oligomers is increased. On the other hand, the P41T mutation destabilizes the disordered structure by populating a heterogeneous distribution of small aggregates, which may be the molecular basis for reduced proteolytic processing by MMP-20 (28).…”

Section: Discussionmentioning

confidence: 99%

Perturbed Amelogenin Secondary Structure Leads to Uncontrolled Aggregation in Amelogenesis Imperfecta Mutant Proteins

Lakshminarayanan¹,

Bromley²,

Lei

et al. 2010

Journal of Biological Chemistry

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Mutations in amelogenin sequence result in defective enamel, and the diverse group of genetically altered conditions is collectively known as amelogenesis imperfecta (AI). Despite numerous studies, the detailed molecular mechanism of defective enamel formation is still unknown. In this study, we have examined the biophysical properties of a recombinant murine amelogenin (rM180) and two point mutations identified from human DNA sequences in two cases of AI (T21I and P41T). At pH 5.8 and 25°C, wild type (WT) rM180 and mutant P41T existed as monomers, and mutant T21I formed lower order oligomers. CD, dynamic light scattering, and fluorescence studies indicated that rM180 and P41T can be classified as a premolten globule-like subclass protein at 25°C. Thermal denaturation and refolding monitored by CD ellipticity at 224 nm indicated the presence of a strong hysteresis in mutants compared with WT. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that WT transformed to a partially folded conformation upon heating and remained stable. The partially folded conformation formed by P41T, however, readily converted into a heterogeneous population of aggregates. T21I existed in an oligomeric state at room temperature and, upon heating, rapidly formed large aggregates over a very narrow temperature range. Thermal denaturation and refolding studies indicated that the mutants are less stable and exhibit poor refolding ability compared with WT rM180. Our results suggest that alterations in self-assembly of amelogenin are a consequence of destabilization of the intrinsic disorder. Therefore, we propose that, like a number of other human diseases, AI appears to be due to the destabilization of the secondary structure as a result of amelogenin mutations.Tooth enamel is one of the hardest and most heavily mineralized vertebrate tissues that can withstand wear without catastrophic failure during the entire life span of an organism (1, 2). The formation of enamel takes place in an extracellular environment, including an array of complex proteins and proteases in three main stages, namely the secretory, transition, and maturation stages (3). As the enamel development progresses, proteases such as enamelysin (MMP-20) and later kallikrein 4 (KLK-4) cleave the proteins, which are removed from the mineralization site in the extracellular matrix enabling the growth of enamel crystals and resulting in enamel hardness. Thus, during amelogenesis, the soft mineralized tissue formed during the early stages becomes hard and tough and almost devoid of any proteins at the maturation stage. Amelogenin is the major constituent (ϳ90%) of the protein matrix during the secretory stage and, together with other proteins in enamel, is responsible for the hierarchical structure observed in the enamel prisms (4 -6).In vitro amelogenin self-assembles into spherical structures, and this property depends on pH, ionic strength, and protein concentration (7). Using CD spectropolarimetry and NMR, we have reported that recomb...

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Section: Discussionmentioning

confidence: 99%

Perturbed Amelogenin Secondary Structure Leads to Uncontrolled Aggregation in Amelogenesis Imperfecta Mutant Proteins

Lakshminarayanan¹,

Bromley²,

Lei

et al. 2010

Journal of Biological Chemistry

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“…The same order applies for the P41T mutant. Recent work on 'amelogenin-to-MMP20' interactions [Tanimoto et al, 2008] also used SPR to calculate relative binding constants. Amelogenin solutions were passed over an immo-bilized layer of deactivated MMP20 and it was found that P41T amelogenin exhibited a lower k (on) signal than did the wild-type rH174 (recombinant human amelogenin).…”

Section: Discussionmentioning

confidence: 99%

Folding, Assembly, and Aggregation of Recombinant Murine Amelogenins with T21I and P41T Point Mutations

Bromley

Lakshminarayanan

Lei

et al. 2011

Cells Tissues Organs

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Two point mutations (T21I and P40T) within amelogenin have been identified from human DNA sequences in 2 instances of amelogenesis imperfecta. We studied the folding and self-assembly of recombinant amelogenin (rM180) compared to the T21I and P40T mutants analogs. At pH 5.8 and 25°C, rM180 and the P41T mutant existed as monomers, whereas the T21I mutant formed small oligomers. At pH 8 and 25°C, all of the amelogenin samples formed nanospheres with hydrodynamic radii (R_H) of around 15–16 nm. Upon heating to 37°C, particles of P41T increased in size (R_H = 18 nm). During thermal denaturation at pH 5.8, both of the mutant proteins refolded more slowly than the wild-type (WT) rM180. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that the WT transformed to a partially folded conformation upon heating and remained stable. Thermal denaturation and refolding studies indicated that the mutants were less stable and exhibit a greater ability to prematurely aggregate compared to the WT. Our data suggest that in the case of P41T, alterations in the self-assembly of amelogenin are a consequence of destabilization of the secondary structure, while in the case of T21I they are a consequence of change in the overall hydrophobicity at the N-terminal region. We propose that alterations in the assembly (i.e. premature aggregation) of mutant amelogenins may have a profound effect on intra- and extracellular processes such as amelogenin secretion, proteolysis, and its interactions with nonamelogenins as well as with the forming mineral.

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“…Hasil penelitian menunjukkan bahwa ekspresi amelogenin kelompok kontrol (K-1) paling rendah dibanding kelompok yang dipajan fluorida yang mempunyai nilai ekspresi paling tinggi. Hal ini disebabkan pada saat proses amelogenesis secara normal, amelogenin akan terdegradasi menjadi beberapa fragmen melalui proses hidrolisis oleh aktivitas Matrixmetalloproteinase-20 (MMP-20) [16][17][18] yang merupakan mediator dari remodeling Gambar 3. A) Gambaran densitas matriks enamel pada kelompok control; B) Gambaran densitas matriks enamel kelompok yang dipajan NaF; C) Gambaran densitas matriks enamel kelompok yang dipajan NaF+NaCl 2 (Pengamatan menggunakan SEM dengan pembesaran 20.000x).…”

Section: Pembahasanunclassified

Peran kalsium sebagai prevensi terjadinya hipoplasia enamel (The role of calcium on enamel hypoplasia prevention)

Wahluyo

2013

Dent. J. (Majalah Kedokteran Gigi)

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Reduced Amelogenin-MMP20 Interactions in Amelogenesis Imperfecta

Cited by 16 publications

References 30 publications

Perturbed Amelogenin Secondary Structure Leads to Uncontrolled Aggregation in Amelogenesis Imperfecta Mutant Proteins

Perturbed Amelogenin Secondary Structure Leads to Uncontrolled Aggregation in Amelogenesis Imperfecta Mutant Proteins

Folding, Assembly, and Aggregation of Recombinant Murine Amelogenins with T21I and P41T Point Mutations

Peran kalsium sebagai prevensi terjadinya hipoplasia enamel (The role of calcium on enamel hypoplasia prevention)

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