Phosphophoryns (PPs), a family of Asp and Ser(P)-rich dentin proteins, are considered to be archetypal regulators of several aspects of extracellular matrix (ECM) biomineralization. We have cloned a rat incisor PP gene, Dmp2, from our odontoblast cDNA library and localized it to mouse chromosome 5q21 within 2 centimorgans of Dmp1, another tooth-specific ECM protein. The phosphophoryns (PPs) 1 (1) are a family of highly phosphorylated proteins with a unique composition and amino acid sequence. Found as the major noncollagenous proteins of the mineralized dentin matrix, the PPs have been considered to be the archetype of macromolecules that might regulate biomineralization processes (2, 3) by binding to the matrix of structural proteins (4), nucleating mineralization (5, 6), and controlling crystal growth (7). To understand how the PPs might carry out these diverse functions, it is crucial to know their amino acid sequences and structures. The PP from various species contain from 35 to 45 residue % aspartic acid and 40 to 55 residue % serine, of which as many as 90% of the Ser residues are phosphorylated (8, 9). The difficulties of Edman sequencing of phosphoserine-rich proteins have limited the information attainable by conventional sequencing efforts (10 -12) to a few internal acidic domains and a short amino-terminal sequence. Recently, chemical sequencing using means to derivatize the phosphoserines has provided a longer NH 2 -terminal sequence (13). Sequencing via cloning of a PP cDNA has also been difficult. A putative PP cDNA was described (14) several years ago, but no sequence data have been reported. Nucleotide probes have generally been unsuccessful because of the high redundancy of the Ser codons (15). Antibody screening of cDNA libraries was not fruitful because of the difficulties of producing high titer PP antibodies (16, 17).We have recently taken a new approach to the production of an antibody to a highly purified, dephosphorylated phosphophoryn (dPP) and, as described below, have been successful in using that antibody to identify several cDNAs in our established rat incisor odontoblast cDNA library (15,18). Sequencing of the clones obtained thus far yielded the expected AspSer-rich composition but, more importantly, revealed both an unexpected, unique "triplet cassette" repeat motif as well as a "doublet" repeat motif. These might account for several of the biomineralization regulatory functions of the phosphophoryns. We report here the nature of these unique sequence blocks. Furthermore, the identification of a PP cDNA clone has allowed the chromosomal localization of the corresponding PP gene. As shown below, the PP gene is colocalized with the gene for a mineralization disorder, dentinogenesis imperfecta, type II, correcting an erroneous assignment (14). EXPERIMENTAL PROCEDURESPP and Antibody Preparation-PP was extracted from rat incisors by the procedure of Rahima and Veis (9). The high M r PP components were isolated by CaCl 2 precipitation from the crude extract, followed by chromatography ...

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Dentin extracellular matrix has been shown to contain components capable of inducing chondrogenesis and osteogenesis at ectopic sites when implanted in vivo, and chondrogenesis in cultures of embryonic muscle-derived fibroblasts (EMF) in vitro. The polypeptide responsible, called the chondrogenic-inducing agent (CIA), has been isolated from a 4.0-M guanidinium hydrochloride extract of demineralized bovine dentin matrix. Following Sephacryl S-100 chromatography, CIA activity was identified in fractions by assay for uptake of [35S]-SO4 into proteoglycan by the EMF after 24 hrs in culture. The active fraction induced the EMF to produce type II collagen mRNA and decrease production of type I collagen mRNA after 5 days in culture. The EMF + CIA, cultured for 4 to 7 wks, formed toluidine-blue- and alizarin-red-stainable nodules, indicative of chondrogenic induction. In vivo implants in rat muscle with collagen carrier produced ectopic bone after 7 wks. The CIA was brought to near-homogeneity by reverse-phase high-performance liquid chromatography, tested at each step by EMF [35S]-SO4-incorporation assays. The CIA components had masses in the ranges of 6000 to 10,000 Da by both mass spectroscopy and gel electrophoresis. The CIA amino acid composition, NH2-terminal, and internal amino acid sequences were determined. These data showed unequivocally that the CIA peptides were derived from bovine amelogenin. The peptides contain the amino-terminal portion of the bovine amelogenin. The presence of these chondrogenic/osteogenic amelogenin-polypeptides in dentin matrix leads us to hypothesize that they may be involved in epithelial-mesenchymal signaling during tooth development interactions-the first time a function has been indicated for these molecules.

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