Boris Sabsay

The Carboxyl-terminal Domain of Phosphophoryn Contains Unique Extended Triplet Amino Acid Repeat Sequences Forming Ordered Carboxyl-Phosphate Interaction Ridges That May Be Essential in the Biomineralization Process

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 carboxyl-terminal region of Dmp2 protein (60 residue % Ser, 31 residue % Asp) is divided into two domains, one with unique repetitive blocks of [DSS]n,3≤14, the other with [SD]m = 2,3. Conformational analysis shows the phosphorylated form of the [DS*S*]n repeats to have a unique structure with well defined ridges of phosphates and carboxyls available for counter ion binding. The [S*D]m domains have different phosphate and carboxylate interaction edges and thus different calcium ion and apatite surface binding properties. These two domains and the colocalization of Dmp1 and Dmp2 genes at a position equivalent to the dentinogenesis imperfecta type II location on human 4q21 all suggest that the PPs are indeed involved in some aspect of ECM mineralization.

Identification of the Chondrogenic-inducing Activity from Bovine Dentin (bCIA) as a Low-molecular-mass Amelogenin Polypeptide

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-redstainable 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, NH2terminal, 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.

Bone and Tooth Formation. Insights into Mineralization Strategies

Mineralized tissues take either of two possible forms, distinguished on the basis of the relationship between mineral and matrix and the organization of the mineral phase. In organisms with shells, carapaces and exoskeletons the mineral crystals are large, form the major continuous phase, and determine the principal physical properties of the tissue. Bone, on the other hand, is a composite-like tissue in which the organic matrix is the continuous phase while the mineral is dispersed as discontinuous microcrystals regularly throughout the matrix. In both types of tissues the organic phase is the cellular product deposited before mineral accretion begins. In both situations it has been postulated that the organic matrix directs the deposition of the mineral phase, from nucleation through ultimate limitation of crystal growth. The central question under investigation here is that of the mechanisms whereby the matrix control of mineralization is expressed. Is there a cannon strategy used in all systems, in spite of the evident diversity?

Characterization of the molecular weights of bovine molar, rat incisor, and other phosphophoryns.

The phosphophoryns show anomalous behavior in solution, and are easily degraded during extraction. They appear in varied forms in the teeth of different species and differ in the teeth of the same species in a developmental and age dependent fashion. This set of properties has made the characterization of the phosphophoryns by biochemical means a difficult and controversial subject. Bovine molar phosphophoryn, bPP, has been characterized in detail by a variety of physical methods, and then compared with the PP from other species by DEAE-HPLC, gel electrophoresis, and immunological cross reactivity. The possible existence of a proPP biosynthetic precursor has been investigated by rat incisor organ culture and examination of the 32P and 14C labeled products. These studies all show marked differences in the Mr values for the PP from teeth of different species, even when they are antigenically cross-reactive.