Phosphorylation of Phosphophoryn Is Crucial for Its Function as a Mediator of Biomineralization

Abstract

Phosphoproteins of the organic matrix of bone and dentin have been implicated as regulators of the nucleation and growth of the inorganic Ca-P crystals of vertebrate bones and teeth. One such protein identified in the dentin matrix is phosphophoryn (PP). It is highly acidic in nature because of a high content of aspartic acid and phosphate groups on serines. The 244-residue carboxyl-terminal domain of rat PP, predominantly containing the aspartic acid-serine repeats, has been cloned, and the corresponding protein has been expressed recombinantly in Escherichia coli. This portion of PP, named DMP2 (dentin matrix protein 2), is not phosphorylated by the bacteria and thus provided a means to study the function of the phosphate groups, the major post-translational modification of native PP. The recombinant DMP2 (rDMP2) possessed much lower calcium binding capacity than native PP. Small angle x-ray scattering experiments demonstrated that PP folds to a compact globular structure upon calcium binding, whereas rDMP2 maintained an unfolded structure. In vitro nucleation experiments showed that PP could nucleate plate-like apatite crystals in pseudophysiological buffer, whereas rDMP2 failed to mediate the transformation of amorphous calcium phosphate to apatite crystals under the same experimental conditions. Collagen binding experiments demonstrated that PP favors the formation of collagen aggregates, whereas in the presence of rDMP2 thin fibrils are formed. Overall these results suggested that the phosphate moieties in phosphophoryn are important for its function as a mediator of dentin biomineralization.Mineralization is an essential requirement for the development of the mechanical properties of hard tissues such as bones and teeth. However, on a volume basis, the mineral constitutes only about 50% of a bone; the remaining extracellular organic matrix (ECM) 2 is a hydrated mixture of collagen and noncollagenous matrix proteins (NCPs). The NCPs constitute 5-10% of the total ECM and are associated with the mineral phase so strongly that the tissues need to be demineralized before those proteins can be extracted. The NCPs are mostly acidic proteins, rich in glutamic acid, aspartic acid, and phosphorylated serine/threonine residues, with a high capacity for binding calcium ions and hydroxyapatite crystal surfaces. Therefore, they have been implicated in the regulation of mineral deposition during osteogenesis and dentinogenesis (1). Phosphorylation of threonine and serine residues takes place as a post-translational modification of the core protein via the action of casein kinases (2). found that dentin mineralization was impaired in the presence of casein kinase inhibitors. Thus post-translational phosphorylation of NCPs is crucial for biomineralization.The major phosphoprotein of dentin is the Asp-and Ser-rich protein called phosphophoryn. The name phosphophoryn (PP) was coined to describe its exceedingly high content of phosphate groups. PP, as isolated from dentin, has a unique composition with aspartyl and seryl ...

Expression of Phosphophoryn Is Sufficient for the Induction of Matrix Mineralization by Mammalian Cells

“…1B). This antibody (a gift from Dr. A. Veis) has been previously shown to have cross-reactivity with phosphophoryn due to the common content of (DSS) n repeats (19,20). We confirmed the correct size and migration of the recombinant PP through Western blot analysis (Fig.…”

“…The precipitated proteins from the medium and cell/matrix were either further purified by column chromatography, or blotted on nitrocellulose paper using dot blot or Western blot techniques using both the anti-PP antibody (generated in our laboratory) and the antidentin matrix protein 2 (DMP2) antibody (a generous gift from Dr. Arthur Veis, Northwestern University). This antibody has been previously shown to have cross-reactivity with phosphophoryn due to the common content of (DSS) n repeats (19,20). As a positive control, we used our recombinant PP as well as recombinant DMP2, provided by Dr. Veis.…”

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

“…We hypothesized that specific proteins were phosphorylated and dephosphorylated during the stages of initiation and proliferation of mineral crystals based on solution free studies of the effects of these proteins [2][3][4][5][6] and on our unpublished micro-array results showing upregulation of kinases during early stages of mineralization and down-regulation of phosphatases at the same time. We also hypothesized that the functions of the phosphatases was not simply to elevate the local inorganic phosphate concentration.…”

“…In cell-free mineralization studies the ability of extracellular matrix phosphoproteins such as osteopontin (OPN) [1,2], bone sialoprotein (BSP) [3,4], dentin matrix protein-1 (DMP1) [5] and phosphophoryn [6] to initiate hydroxyapatite (HA) mineral deposition and regulate the rate and extent of growth of the HA crystals is dependent on their state of phosphorylation. While the importance of the phosphate ion for appropriate mineral deposition is well recognized [7], it is only in recent years that attention has been paid to whether or not the extracellular matrix proteins were phosphorylated, as well as to their extent of phosphorylation and the enzymes involved in these posttranslational modifications [1][2][3][4][5][6][7][8][9].…”

“…While the importance of the phosphate ion for appropriate mineral deposition is well recognized [7], it is only in recent years that attention has been paid to whether or not the extracellular matrix proteins were phosphorylated, as well as to their extent of phosphorylation and the enzymes involved in these posttranslational modifications [1][2][3][4][5][6][7][8][9]. The importance of phosphorylation and dephosphorylation has been suggested by the abnormal patterns of mineralization in mutant animals with defective phosphoprotein degradative enzymes [10][11][12], defective phosphate transport [13,14], or defective phosphorylation [15].…”