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Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus.

ABSTRACT: We have recently reported that the extracellular enamel protein amelogenin has affinity to interact with phospholipids and proposed that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. Here, in order to identify the liposome-interacting domains of amelogenin we designed four different amelogenin mutants containing only a single tryptophan at positions 25, 45, 112 and 161. Circular dichroism studies of the mutants confirmed that they are structurally similar to the wild-type amelogenin. Utilizing the intrinsic fluorescence of single tryptophan residue and fluorescence resonance energy transfer [FRET], we analyzed the accessibility and strength of their binding with an ameloblast cell membrane-mimicking model membrane (ACML) and a negatively charged liposome used as a membrane model. We found that amelogenin has membrane-binding ability mainly via its N-terminal, close to residues W25 and W45. Significant blue shift was also observed in the fluorescence of a N-terminal peptide following addition of liposomes. We suggest that, among other mechanisms, enamel malformation in cases of Amelogenesis Imperfecta (AI) with mutations at the N-terminal may be the result of defective amelogenin-cell interactions.

SUBMITTER: Lokappa SB

PROVIDER: S-EPMC4532586 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus.

Lokappa Sowmya Bekshe SB Chandrababu Karthik Balakrishna KB Moradian-Oldak Janet J

Biochemical and biophysical research communications 20150717 3

We have recently reported that the extracellular enamel protein amelogenin has affinity to interact with phospholipids and proposed that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. Here, in order to identify the liposome-interacting domains of amelogenin we designed four different amelogenin mutants containing only a single tryptophan at positions 25, 45, 112 and 161. Circular dichroism studies of the mutants confirmed tha ...[more]

PMID: 26188506

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Project description:BACKGROUND:Polarity is necessary for epithelial cells to perform distinct functions at their apical and basal surfaces. Oral epithelial cell-derived ameloblasts at secretory stage (SABs) synthesize large amounts of enamel matrix proteins (EMPs), largely amelogenins. EMPs are unidirectionally secreted into the enamel space through their apical cytoplasmic protrusions, or Tomes' processes (TPs), to guide the enamel formation. Little is known about the transcriptional regulation underlying the establishment of cell polarity and unidirectional secretion of SABs. RESULTS:The higher-order chromatin architecture of eukaryotic genome plays important roles in cell- and stage-specific transcriptional programming. A genome organizer, special AT-rich sequence-binding protein 1 (SATB1), was discovered to be significantly upregulated in ameloblasts compared to oral epithelial cells using a whole-transcript microarray analysis. The Satb1-/- mice possessed deformed ameloblasts and a thin layer of hypomineralized and non-prismatic enamel. Remarkably, Satb1-/- ameloblasts at the secretory stage lost many morphological characteristics found at the apical surface of wild-type (wt) SABs, including the loss of Tomes' processes, defective inter-ameloblastic adhesion, and filamentous actin architecture. As expected, the secretory function of Satb1-/- SABs was compromised as amelogenins were largely retained in cells. We found the expression of epidermal growth factor receptor pathway substrate 8 (Eps8), a known regulator for actin filament assembly and small intestinal epithelial cytoplasmic protrusion formation, to be SATB1 dependent. In contrast to wt SABs, EPS8 could not be detected at the apical surface of Satb1-/- SABs. Eps8 expression was greatly reduced in small intestinal epithelial cells in Satb1-/- mice as well, displaying defective intestinal microvilli. CONCLUSIONS:Our data show that SATB1 is essential for establishing secretory ameloblast cell polarity and for EMP secretion. In line with the deformed apical architecture, amelogenin transport to the apical secretory front and secretion into enamel space were impeded in Satb1-/- SABs resulting in a massive cytoplasmic accumulation of amelogenins and a thin layer of hypomineralized enamel. Our studies strongly suggest that SATB1-dependent Eps8 expression plays a critical role in cytoplasmic protrusion formation in both SABs and in small intestines. This study demonstrates the role of SATB1 in the regulation of amelogenesis and the potential application of SATB1 in ameloblast/enamel regeneration.

Project description:Tooth enamel forms in an ephemeral protein matrix where changes in protein abundance, composition and posttranslational modifications are critical to achieve healthy enamel properties. Amelogenin (AMELX) with its splice variants is the most abundant enamel matrix protein, with only one known phosphorylation site at serine 16 shown in vitro to be critical for regulating mineralization. The phosphorylated form of AMELX stabilizes amorphous calcium phosphate, while crystalline hydroxyapatite forms in the presence of the unphosphorylated protein. While AMELX regulates mineral transitions over space and time, it is unknown whether and when un-phosphorylated amelogenin occurs during enamel mineralization. This study aims to reveal the spatiotemporal distribution of the cleavage products of the most abundant AMLEX splice variants including the full length P173, the shorter leucine-rich amelogenin protein (LRAP), and the exon 4-containing P190 in forming enamel, all within the context of the changing enamel matrix proteome during mineralization. We microsampled permanent pig molars, capturing known stages of enamel formation from both crown surface and inner enamel. Nano-LC-MS/MS proteomic analyses after tryptic digestion rendered more than 500 unique protein identifications in enamel, dentin, and bone. We mapped collagens, keratins, and proteolytic enzymes (CTSL, MMP2, MMP10) and determined distributions of P173, LRAP, and P190 products, the enamel proteins enamelin (ENAM) and ameloblastin (AMBN), and matrix-metalloprotease-20 (MMP20) and kallikrein-4 (KLK4). All enamel proteins and KLK4 were near-exclusive to enamel and in excellent agreement with published abundance levels. Phosphorylated P173 and LRAP products decreased in abundance from recently deposited matrix toward older enamel, mirrored by increasing abundances of testicular acid phosphatase (ACPT). Our results showed that hierarchical clustering analysis of secretory enamel links closely matching distributions of unphosphorylated P173 and LRAP products with ACPT and non-traditional amelogenesis proteins, many associated with enamel defects. We report higher protein diversity than previously published and Gene Ontology (GO)-defined protein functions related to the regulation of mineral formation in secretory enamel (e.g., casein α-S1, CSN1S1), immune response in erupted enamel (e.g., peptidoglycan recognition protein, PGRP), and phosphorylation. This study presents a novel approach to characterize and study functional relationships through spatiotemporal mapping of the ephemeral extracellular matrix proteome.

Dataset Information

Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus.

Publications

Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus.

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