The α-chains of the collagen superfamily are encoded with information that specifies self-assembly into fibrils, microfibrils, and networks that have diverse functions in the extracellular matrix. A ...key self-organizing step, common to all collagen types, is trimerization that selects, binds, and registers cognate α-chains for assembly of triple helical protomers that subsequently oligomerize into specific suprastructures. In this article, we review recent findings on the mechanism of chain selection and infer that terminal noncollagenous domains function as recognition modules in trimerization and are therefore key determinants of specificity in the assembly of suprastructures. This mechanism is also illustrated with computer-generated animations.
Abstract
Biomineralization is the process by which living organisms deposit mineral in the extracellular matrix. In nature, almost 50% of biominerals are calcium-bearing minerals. In addition to ...calcium, we find biominerals formed from silica and magnetite. Calcium-containing biominerals could be either calcium phosphate as in apatite found in vertebrates or calcium carbonate as in calcite and aragonite found in many invertebrates. Since all biomineralization is matrix mediated, an understanding of the nature of the proteins involved is essential in elucidating its mechanism. This review will discuss some of the proteins involved in the process of biomineralization involving calcium. Two proteins, dentin matrix protein 1 and dentin phosphoprotein (Phosphophoryn) will serve as models for the vertebrate system, and two others - P16 and phosphodontin will serve as models for the invertebrate system.
Dentin phosphoprotein (DPP) is the most abundant noncollagenous protein in the dentin, where it plays a major role in the mineralization of dentin. However, we and others have shown that in addition ...to being present in the dentin, DPP is also present in nonmineralizing tissues like the kidney, lung, and salivary glands, where it conceivably has other functions such as in calcium transport. Because annexins have been implicated as calcium transporters, we examined the relationships between DPP and annexins. In this report, we show that DPP binds to annexin 2 and 6 present in a rat ureteric bud cell line (RUB1). Immunofluorescence studies show that annexin 2 and DPP colocalize in these cells. In addition, DPP and annexin 2 colocalize in the ureteric bud branches of embryonic metanephric kidney. In the RUB1 cells and ureteric bud branches of embryonic kidney, colocalization was restricted to the cell membrane. Studies on calcium influx into RUB cells show that in the presence of anti-DPP, there was a 40% reduction of calcium influx into these cells. We postulate that DPP has different functions in the kidney as compared with the odontoblasts. In the odontoblasts, its primary function is in the extracellular mineralization of dentin, whereas in the kidney it may participate in calcium transport.
Background: Dentin phosphoprotein (DPP), responsible for tooth mineralization, is also present in nonmineralizing tissues like kidney, lung, and salivary glands.
Results: DPP binds annexin 2 and 6 and plays a role in calcium influx in rat ureteric bud cells.
Conclusion: DPP plays different roles in mineralizing and nonmineralizing tissues.
Significance: This is the first report showing a possible function for DPP in nonmineralizing tissues.
Studies of mineralization of embryonic spicules and of the sea urchin genome have identified several putative mineralization-related proteins. These predicted proteins have not been isolated or ...confirmed in mature mineralized tissues. Mature Lytechinus variegatus teeth were demineralized with 0.6 n HCl after prior removal of non-mineralized constituents with 4.0 m guanidinium HCl. The HCl-extracted proteins were fractionated on ceramic hydroxyapatite and separated into bound and unbound pools. Gel electrophoresis compared the protein distributions. The differentially present bands were purified and digested with trypsin, and the tryptic peptides were separated by high pressure liquid chromatography. NH2-terminal sequences were determined by Edman degradation and compared with the genomic sequence bank data. Two of the putative mineralization-related proteins were found. Their complete amino acid sequences were cloned from our L. variegatus cDNA library. Apatite-binding UTMP16 was found to be present in two isoforms; both isoforms had a signal sequence, a Ser-Asp-rich extracellular matrix domain, and a transmembrane and cytosolic insertion sequence. UTMP19, although rich in Glu and Thr did not bind to apatite. It had neither signal peptide nor transmembrane domain but did have typical nuclear localization and nuclear exit signal sequences. Both proteins were phosphorylated and good substrates for phosphatase. Immunolocalization studies with anti-UTMP16 show it to concentrate at the syncytial membranes in contact with the mineral. On the basis of our TOF-SIMS analyses of magnesium ion and Asp mapping of the mineral phase composition, we speculate that UTMP16 may be important in establishing the high magnesium columns that fuse the calcite plates together to enhance the mechanical strength of the mineralized tooth.
Embryonic mouse tooth germs were cultured in vitro in the presence of two related amelogenin isoforms to determine their effects on tooth development. Our results show that these individual proteins ...have specific but quite different effects on epithelial‐derived ameloblasts versus mesenchymal‐derived odontoblasts.
Introduction: Amelogenins, the main protein components of enamel matrix, have been shown to have signaling activity. Amelogenin isoforms differing only by the presence or exclusion of exon 4, designated ‘A+4’ (composed of exons 2, 3, 4, 5, 6d, and 7) and ‘A‐4’ (composed of exons 2, 3, 4, 5, 6d, and 7) and ‘A‐4’ (composed of exons 2, 3, 5, 6d, and 7), showed similar, but different, effects both in vitro and in vivo on postnatal teeth.
Materials and Methods: Lower first molar tooth germs of E15/16 CD1 mice were microdissected and cultured in vitro in a semisolid media containing either 20% FBS, 2% FBS, or 2% FBS with either 1.5 nM ‘A+4’, ‘A‐4’, or both for 6 days. Tooth germs were analyzed by H&E staining and immunohistochemistry for collagen I, dentin matrix protein 2, and DAPI nuclear staining.
Results: Teeth cultured in media containing 20% FBS showed normal development with polarized ameloblasts, and odontoblasts producing dentin matrix, and DMP2 expression in odontoblasts and pre‐ameloblasts. Culture in 2% FBS media resulted in no ameloblast polarization and modest odontoblast differentiation with scant dentin matrix. Tooth germs cultured with ‘A+4’ in 2% FBS media had well‐polarized odontoblasts with robust dentin production and concomitant ameloblast polarization. DMP2 expression was equal to or greater than seen in the 20% FBS culture condition. In cultures with ‘A‐4’ in 2% FBS media, odontoblast polarization and dentin production was reduced compared with ‘A+4’. However, the pre‐ameloblast layer was disorganized, with no ameloblast polarization occurring along the dentin surface. DMP2 expression was reduced in the odontoblasts compared with the 20% FBS and ‘A+4’ conditions and was almost completely abrogated in the pre‐ameloblasts.
Conclusion: These data show different signaling activities of these closely related amelogenin isoforms on tooth development. Here we make the novel observation that ‘A−4’ has an inhibitory effect on ameloblast development, whereas ‘A+4’ strongly stimulates odontoblast development. We show for the first time that specific amelogenin isoforms have effects on embryonic tooth development in vitro and also hypothesize that DMP2 may play a role in the terminal differentiation of both ameloblasts and odontoblasts.
Low molecular mass amelogenin-related polypeptides extracted from mineralized dentin have the ability to affect the differentiation pathway of embryonic muscle fibroblasts in culture and lead to the ...formation of mineralized matrix in in vivo implants. The objective of the present study was to determine whether the bioactive peptides could have been amelogenin protein degradation products or specific amelogenin gene splice products. Thus, the splice products were prepared, and their activities were determined in vitro and in vivo. A rat incisor tooth odontoblast pulp cDNA library was screened using probes based on the peptide amino acid sequencing data. Two specific cDNAs comprised from amelogenin gene exons 2,3,4,5,6d,7 and 2,3,5,6d,7 were identified. The corresponding recombinant proteins, designated rA+4 (8.1 kDa) and rA−4 (6.9 kDa), were produced. Both peptides enhanced in vitrosulfate incorporation into proteoglycan, the induction of type II collagen, and Sox9 or Cbfa1 mRNA expression. In vivoimplant assays demonstrated implant mineralization accompanied by vascularization and the presence of the bone matrix proteins, BSP and BAG-75. We postulate that during tooth development these specific amelogenin gene splice products, A+4 and A−4, may have a role in preodontoblast maturation. The A+4 and A−4 may thus be tissue-specific epithelial mesenchymal signaling molecules.
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