Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development

Abstract

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8–16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit.

A comparison of proteomic, genomic, and osteological methods of archaeological sex estimation

“…Proteomic sex estimation exploits the fact that the highly characterized sex-chromosome-specific amelogenin gene family is expressed as proteins in the most robust tissue in the human body, enamel 33 , 34 , 37 , 58 , 71 . The proteins are cleaved into peptides in situ, as part of enamel formation during tooth biogenesis 50 , 72 , 73 . In order to extract and analyze this peptide population, researchers need to demineralize the enamel and most use acid-based 33 – 36 , 45 , 46 , 55 , 58 , 74 approaches.…”

Enamel biomimetics—fiction or future of dentistry

“…31 This process is based on a polysaccharide/bisphosphonate matrix (chitosan/alendronate) and mimics key steps of initial amelogenesis, including (i) formation of a Ca/P-rich amelogenin protein matrix, (ii) enzymatic degradation and continued crystal growth, and (iii) crystal elongation as facilitated by elongated amelogenin fragments. 32 In their paper, the authors suggested that a combination of non-classical crystallization mechanisms, development of synthetic amelogenin analogues, and imitating remaining biomineralization steps would hold great promise for future approaches to improve the repair of enamel defects. 31…”

Controlling Enamel Remineralization by Amyloid‐Like Amelogenin Mimics

“…Once secreted into the extracellular space, amelogenin undergoes a series of proteolytic cleavages starting at the C‐Ame and assemble into nanospheres with a diameter of ≈10–20 nm, which are believed to play a key role in the mineralization and structural organization of enamel. [ 7–9 ] The N‐Ame is crucial for amelogenin self‐assembly into amyloid‐like aggregation through β‐sheet stacking to template the growth of HAp crystals, [ 10,11 ] and the C‐Ame mediate parallel alignment of these crystals [ 12–14 ] ( Scheme ). Hierarchically organized HAp nanorods are further formed in nature involving an aggregation of amorphous calcium phosphate (ACP) nanospheres and a transformation of ACP to HAp crystals (Figure S1, Supporting Information).…”