SM50 repeat-polypeptides self-assemble into discrete matrix subunits and promote appositional calcium carbonate crystal growth during sea urchin tooth biomineralization.

The two major proteins involved in vertebrate enamel formation and echinoderm sea urchin tooth biomineralization, amelogenin and SM50, are both characterized by elongated polyproline repeat domains in the center of the macromolecule. To determine the role of polyproline repeat polypeptides in basal deuterostome biomineralization, we have mapped the localization of SM50 as it relates to crystal growth, conducted self-assembly studies of SM50 repeat polypeptides, and examined their effect on calcium carbonate and apatite crystal growth. Electron micrographs of the growth zone of Strongylocentrotus purpuratus sea urchin teeth documented a series of successive events from intravesicular mineral nucleation to mineral deposition at the interface between tooth surface and odontoblast syncytium. Using immunohistochemistry, SM50 was detected within the cytoplasm of cells associated with the developing tooth mineral, at the mineral secreting front, and adjacent to initial mineral deposits, but not in muscles and ligaments. Polypeptides derived from the SM50 polyproline alternating hexa- and hepta-peptide repeat region (SM50P6P7) formed highly discrete, donut-shaped self-assembly patterns. In calcium carbonate crystal growth studies, SM50P6P7 repeat peptides triggered the growth of expansive networks of fused calcium carbonate crystals while in apatite growth studies, SM50P6P7 peptides facilitated the growth of needle-shaped and parallel arranged crystals resembling those found in developing vertebrate enamel. In comparison, SM50P6P7 surpassed the PXX24 polypeptide repeat region derived from the vertebrate enamel protein amelogenin in its ability to promote crystal nucleation and appositional crystal growth. Together, these studies establish the SM50P6P7 polyproline repeat region as a potent regulator in the protein-guided appositional crystal growth that occurs during continuous tooth mineralization and eruption. In addition, our studies highlight the role of species-specific polyproline repeat motifs in the formation of discrete self-assembled matrices and the resulting control of mineral growth.

Membranes, minerals, and proteins of developing vertebrate enamel.

Developing tooth enamel is formed as organized mineral in a specialized protein matrix. In order to analyze patterns of enamel mineralization and enamel protein expression in species representative of the main extant vertebrate lineages, we investigated developing teeth in a chondrichthyan, the horn shark, a teleost, the guppy, a urodele amphibian, the Mexican axolotl, an anuran amphibian, the leopard frog, two lepidosauria, a gecko and an iguana, and two mammals, a marsupial, the South American short-tailed gray opossum, and the house mouse. Electron microscopic analysis documented the presence of a distinct basal lamina in all species investigated. Subsequent stages of enamel biomineralization featured highly organized long and parallel enamel crystals in mammals, lepidosaurians, the frog, and the shark, while amorphous mineral deposits and/or randomly oriented crystals were observed in the guppy and the axolotl. In situ hybridization using a full-length mouse probe for amelogenin mRNA resulted in amelogenin specific signals in mouse, opossum, gecko, frog, axolotl, and shark. Using immunohistochemistry, amelogenin and tuftelin enamel proteins were detected in the enamel organ of many species investigated, but tuftelin epitopes were also found in other tissues. The anti-M179 antibody, however, did not react with the guppy and axolotl enameloid matrix. We conclude that basic features of vertebrate enamel/enameloid formation such as the presence of enamel proteins or the mineral deposition along the dentin-enamel junction were highly conserved in vertebrates. There were also differences in terms of enamel protein distribution and mineral organization between the vertebrates lineages. Our findings indicated a correlation between the presence of amelogenins and the presence of long and parallel hydroxyapatite crystals in tetrapods and shark.

Conservation and variation in enamel protein distribution during vertebrate tooth development.

Vertebrate enamel formation is a unique synthesis of the function of highly specialized enamel proteins and their effect on the growth and organization of apatite crystals. Among tetrapods, the physical structure of enamel is highly conserved, while there is a greater variety of enameloid tooth coverings in fish. In the present study, we postulated that in enamel microstructures of similar organization, the principle components of the enamel protein matrix would have to be highly conserved. In order to identify the enamel proteins that might be most highly conserved and thus potentially most essential to the process of mammalian enamel formation, we used immunoscreening with enamel protein antibodies as a means to assay for degrees of homology to mammalian enamel proteins. Enamel preparations from mouse, gecko, frog, lungfish, and shark were screened with mammalian enamel protein antibodies, including amelogenin, enamelin, tuftelin, MMP20, and EMSP1. Our results demonstrated that amelogenin was the most highly conserved enamel protein associated with the enamel organ, enamelin featured a distinct presence in shark enameloid but was also present in the enamel organ of other species, while the other enamel proteins, tuftelin, MMP20, and EMSP1, were detected in both in the enamel organ and in other tissues of all species investigated. We thus conclude that the investigated enamel proteins, amelogenin, enamelin, tuftelin, MMP20, and EMSP1, were highly conserved in a variety of vertebrate species. We speculate that there might be a unique correlation between amelogenin-rich tetrapod and lungfish enamel with long and parallel crystals and enamelin-rich basal vertebrate enameloid with diverse patterns of crystal organization.