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Partial high-resolution structure of phosphorylated and non-phosphorylated leucine-rich amelogenin protein adsorbed to hydroxyapatite.

ABSTRACT: The formation of biogenic materials requires the interaction of organic molecules with the mineral phase. In forming enamel, the amelogenin proteins contribute to the mineralization of hydroxyapatite (HAp). Leucine-rich amelogenin protein (LRAP) is a naturally occurring splice variant of amelogenin that comprises amelogenin's predicted HAp binding domains. We determined the partial structure of phosphorylated and non-phosphorylated LRAP variants bound to HAp using combined solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. New ssNMR measurements in the N-terminus indicate a largely extended structure for both variants, though some measurements are consistent with a partially helical N-terminal segment. The N-terminus of the phosphorylated variant is found to be consistently closer to the HAp surface than the non-phosphorylated variant. Structure prediction was biased using 21 ssNMR measurements in the N- and C-terminus at five HAp crystal faces. The predicted fold of LRAP is similar at all HAp faces studied, regardless of phosphorylation. Largely consistent with experimental observations, LRAP's predicted structure is relatively extended with a helix-turn-helix motif in the N-terminal domain and some helix in the C-terminal domain, and the N-terminal domain of the phosphorylated variant binds HAp more closely than the N-terminal domain of the non-phosphorylated variant. Predictions for both variants show some potential binding specificity for the {010} HAp crystal face, providing further support that amelogenins block crystal growth on the a and b faces to allow elongated crystals in the c-axis.

SUBMITTER: Masica DL

PROVIDER: S-EPMC3155182 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Partial high-resolution structure of phosphorylated and non-phosphorylated leucine-rich amelogenin protein adsorbed to hydroxyapatite.

Masica David L DL Gray Jeffrey J JJ Shaw Wendy J WJ

The journal of physical chemistry. C, Nanomaterials and interfaces 20110701 28

The formation of biogenic materials requires the interaction of organic molecules with the mineral phase. In forming enamel, the amelogenin proteins contribute to the mineralization of hydroxyapatite (HAp). Leucine-rich amelogenin protein (LRAP) is a naturally occurring splice variant of amelogenin that comprises amelogenin's predicted HAp binding domains. We determined the partial structure of phosphorylated and non-phosphorylated LRAP variants bound to HAp using combined solid-state NMR (ssNMR ...[more]

PMID: 21845207

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Project description:We report here a novel biomimetic approach to the regeneration of human enamel. The approach combines the use of inorganic pyrophosphate (PPi) to control the onset and rate of enamel regeneration and the use of leucine-rich amelogenin peptide (LRAP), a nonphosphorylated 56-amino acid alternative splice product of amelogenin, to regulate the shape and orientation of growing enamel crystals. This study builds on our previous findings that show LRAP can effectively guide the formation of ordered arrays of needle-like hydroxyapatite (HA) crystals in vitro and on the known role mineralization inhibitors, like PPi, play in the regulation of mineralized tissue formation. Acid-etched enamel surfaces of extracted human molars, cut perpendicular or parallel to the direction of the enamel rods, were exposed to a PPi-stabilized supersaturated calcium phosphate (CaP) solution containing 0 to 0.06 mg/mL LRAP for 20 h. In the absence of LRAP, PPi inhibition was reversed by the presence of etched enamel surfaces and led to the formation of large, randomly distributed plate-like HA crystals that were weakly attached, regardless of rod orientation. In the presence of 0.04 mg/mL LRAP, however, densely packed mineral layers, comprising bundles of small needle-like HA crystals, formed on etched surfaces that were cut perpendicular to the enamel rods. These crystals were strongly attached, and their arrangement reflected to a significant degree the underlying enamel prism pattern. In contrast, under the same conditions with LRAP, little to no crystal formation was found on enamel surfaces that were cut parallel to the direction of the enamel rods. These results suggest that LRAP preferentially interacts with ab surfaces of mature enamel crystals, inhibiting their directional growth, thus selectively promoting linear growth along the c-axis of enamel crystals. The present findings demonstrate a potential for the development of a new approach to regenerate enamel structure and properties.

Project description:The interactions between proteins and surfaces are critical to a number of important processes including biomineralization, the biocompatibility of biomaterials, and the function of biosensors. Although many proteins exist as monomers or small oligomers, amelogenin is a unique protein that self-assembles into supramolecular structures called "nanospheres," aggregates of hundreds of monomers that are 20-60 nm in diameter. The nanosphere quaternary structure is observed in solution; however, the quaternary structure of amelogenin adsorbed onto hydroxyapatite (HAP) surfaces is not known even though it may be important to amelogenin's function in forming highly elongated and intricately assembled HAP crystallites during enamel formation. We report studies of the interactions of the enamel protein, amelogenin (rpM179), with a well-defined (100) face prepared by the synthesis of large crystals of HAP. High-resolution in situ atomic force microscopy (AFM) was used to directly observe protein adsorption onto HAP at the molecular level within an aqueous solution environment. Our study shows that the amelogenin nanospheres disassemble onto the HAP surface, breaking down into oligomeric (25-mer) subunits of the larger nanosphere. In some cases, the disassembly event is directly observed by in situ imaging for the first time. Quantification of the adsorbate amounts by size analysis led to the determination of a protein binding energy (17.1k(b)T) to a specific face of HAP (100). The kinetics of disassembly are greatly slowed in aged solutions, indicating that there are time-dependent increases in oligomer-oligomer binding interactions within the nanosphere. A small change in the sequence of amelogenin by the attachment of a histidine tag to the N-terminus of rpM179 to form rp(H)M180 results in the adsorption of a complete second layer on top of the underlying first layer. Our research elucidates how supramolecular protein structures interact and break down at surfaces and how small changes in the primary sequence of amelogenin can affect the disassembly process.

Project description:Amelogenins, major extra cellular matrix proteins of developing tooth enamel, are predominantly expressed by ameloblasts and play significant roles in the formation of enamel. Recently, amelogenin has been detected in various epithelial and mesenchymal tissues, implicating that it might have distinct functions in various tissues. We have previously reported that leucine rich amelogenin peptide (LRAP), one of the alternate splice forms of amelogenin, regulates receptor activator of NF-kappa B ligand (RANKL) expression in cementoblast/periodontal ligament cells, suggesting that the amelogenins, especially LRAP, might function as a signaling molecule in bone metabolism. The objective of this study was to identify and define LRAP functions in bone turnover. We engineered transgenic (TgLRAP) mice using a murine 2.3kb α1(I)-collagen promoter to drive expression of a transgene consisting of LRAP, an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) to study functions of LRAP in bone formation and resorption. Calvarial cell cultures from the TgLRAP mice showed increased alkaline phosphatase (ALP) activity and increased formation of mineralized nodules compared to the cells derived from wild-type (WT) mice. The TgLRAP calvarial cells also showed an inhibitory effect on osteoclastogenesis in vitro. Gene expression comparison by quantitative polymerase chain reaction (Q-PCR) in calvarial cells indicated that bone formation makers such as Runx2, Alp, and osteocalcin were increased in TgLRAP compared to the WT cells. Meanwhile, Rankl expression was decreased in the TgLRAP cells in vitro. The ovariectomized (OVX) TgLRAP mice resisted bone loss induced by ovariectomy resulting in higher bone mineral density in comparison to OVX WT mice. The quantitative analysis of calcein intakes indicated that the ovariectomy resulted in increased bone formation in both WT and TgLRAP mice; OVX TgLRAP appeared to show the most remarkably increased bone formation. The parameters for bone resorption in tissue sections showed increased number of osteoclasts in OVX WT, but not in OVX TgLRAP over that of sham operated WT or TgLRAP mice, supporting the observed bone phenotypes in OVX mice. This is the first report identifying that LRAP, one of the amelogenin splice variants, affects bone turnover in vivo.

Dataset Information

Partial high-resolution structure of phosphorylated and non-phosphorylated leucine-rich amelogenin protein adsorbed to hydroxyapatite.

Publications

Partial high-resolution structure of phosphorylated and non-phosphorylated leucine-rich amelogenin protein adsorbed to hydroxyapatite.

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