The SIBLING protein family is a group of non-collagenous proteins (NCPs) that includes dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), and osteopontin (OPN). In the present study, we compared these four proteins in different phases of rat dentin and bone. First, we extracted NCPs in the unmineralized matrices and cellular compartments using guanidium-HCl (G1). Second, we extracted NCPs closely associated with hydroxyapatite using an EDTA solution (E). Last, we extracted the remaining NCPs again with guanidium-HCl (G2). Each fraction of Q-Sepharose ion-exchange chromatography was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Stains-All stain, and with western immunoblotting. In dentin, the NH(2)-terminal fragment of DSPP and its proteoglycan form were primarily present in the G1 extract, whereas the COOH-terminal fragment of DSPP was present exclusively in the E extract. The processed NH(2)-terminal fragment of DMP1 was present in G1 and E extracts, whereas the COOH-terminal fragment of DMP1 existed mainly in the E extract. Bone sialoprotein was present in all three extracts of dentin and bone, whereas OPN was present only in the G1 and E extracts of bone. The difference in the distribution of the SIBLING proteins between organic and inorganic phases supports the belief that these molecular species play different roles in dentinogenesis and osteogenesis.

Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein shown by gene ablations to be critical for the proper mineralization of bone and dentin. In the extracellular matrix of these tissues DMP1 is present as fragments representing the NH 2 -terminal (37 kDa) and COOH-terminal (57 kDa) portions of the cDNA-deduced amino acid sequence. During our separation of bone noncollagenous proteins, we observed a high molecular weight, DMP1-related component (designated DMP1-PG). We purified DMP1-PG with a monoclonal anti-DMP1 antibody affinity column. Amino acid analysis and Edman degradation of tryptic peptides proved that the core protein for DMP1-PG is the 37-kDa fragment of DMP1. Chondroitinase treatments demonstrated that the slower migration rate of DMP1-PG is due to the presence of glycosaminoglycan. Quantitative disaccharide analysis indicated that the glycosaminoglycan is made predominantly of chondroitin 4-sulfate. Further analysis on tryptic peptides led us to conclude that a single glycosaminoglycan chain is linked to the core protein via Ser 74 , located in the Ser 74 -Gly 75 dipeptide, an amino acid sequence specific for the attachment of glycosaminoglycans. Our findings show that in addition to its existence as a phosphoprotein, the NH 2 -terminal fragment from DMP1 occurs as a proteoglycan. Amino acid sequence alignment analysis showed that the Ser 74 -Gly 75 dipeptide and its flanking regions are highly conserved among a wide range of species from caiman to the Homo sapiens, indicating that this glycosaminoglycan attachment domain has survived an extremely long period of evolution pressure, suggesting that the glycosaminoglycan may be critical for the basic biological functions of DMP1.Dentin matrix protein 1 (DMP1), 2 first identified by cDNA cloning using a rat odontoblast mRNA library (1), is a member of the SIBLING protein family (2). Although originally postulated to be dentin-specific, DMP1 expression was later detected in bone (3), brain (4), salivary gland (5), and kidney (6). The cDNA from a number of species has been cloned and sequenced, including that from rat (1), mouse (3), bovine (4), human (7), and chicken (8). The characteristic feature of DMP1 is that it contains an unusually large number of acidic domains, a property that would be consistent with a role in regulating matrix mineralization (1). This purported biological function is supported by observations that transgenic MC3T3-E1 cells overexpressing DMP1 demonstrated higher levels of in vitro mineralization (9). Findings from gene knock-out mice further indicate a role for DMP1 in mineralization; mice lacking the Dmp1 gene demonstrate profound defects in bone and dentin mineralization (10, 11). The observation that the expression of DMP1 in osteocytes is elevated by mechanical stress suggests that this molecule may be involved in the mechanical transduction pathways (12).Other in vitro studies indicate that DMP1 may have non-mineralization-related functions. DMP1 secreted into the blood binds Factor H and blocks the alterna...

Dentin matrix protein 1 (DMP1) has been identified in the extracellular matrix (ECM) of dentin and bone as the processed NH 2 -terminal and COOH-terminal fragment. However, the full-length form of DMP1 has not been identified in these tissues. The focus of this investigation was to search for the intact full-length DMP1 in dentin and bone. We used two types of anti-DMP1 antibodies to identify DMP1: one type specifically recognizes the NH 2 -terminal region and the other type is only reactive to the COOH-terminal region of the DMP1 amino acid sequence. An ~105-kDa protein, extracted from the ECM of rat dentin and bone, was recognized by both types of antibodies; and the migration rate of this protein was identical to the recombinant mouse full-length DMP1 made in eukaryotic cells. We concluded that this ~105-kDa protein is the full-length form of DMP1, which is considerably less abundant than its processed fragments in the ECM of dentin and bone. We also detected the full-length form of DMP1 and its processed fragments in the extract of dental pulp/ odontoblast complex dissected from rat teeth. In addition, immunofluorescence analysis showed that in MC3T3-E1 cells the NH 2 -terminal and COOH-terminal fragments of DMP1 are distributed differently. Our findings indicate that the majority of DMP1 must be cleaved within the cells that synthesize it and that minor amounts of uncleaved DMP1 molecules are secreted into the ECM of dentin and bone. KeywordsDentin matrix protein 1; Posttranslational modification; Extracellular matrix; Dentin; Bone Dentin matrix protein 1 (DMP1), discovered by cDNA cloning, was originally postulated to be dentin-specific [1]. Later on, its expression was observed in bone [2,3]. The distinctive feature of DMP1 is the presence of a large number of acidic domains, a property that implicates it as a possible participant in regulating matrix mineralization. This purported biological function is supported by observations that MC3T3-E1 cells overexpressing DMP1 demonstrate an earlier onset of mineralization and the formation of a significantly larger size of the induced mineralized nodules compared to nontransfected control cells [4]. Findings from Dmp1 knockout mouse experiments and gene mutation studies on human osteomalacia strengthen the conclusion that DMP1 plays an important role in bone and dentin mineralization [5][6][7]. In addition to its direct role in biomineralization, studies indicated that DMP1 may regulate osteoblast-specific and/or odontoblast-specific genes [8,9]. More recent studies indicated that DMP1 may also be involved in the regulation of phosphate homeostasis through fibroblast growth factor 23 (FGF23), a newly identified hormone that is released from bone and targeted in the kidneys; deletion of the Dmp1 gene leads to a dramatic increase of FGF23 mRNA in osteocytes [7].Full-length DMP1 cDNA from a number of species has been cloned and sequenced [1,2,3,10,11], but the corresponding full-length form of the protein has not been identified. In searching for naturally ...

Dentin matrix protein 1 (DMP1) has been shown to be critical for the formation of dentin and bone. However, the precise pathway by which DMP1 participates in dentinogenesis and osteogenesis remains to be clarified. DMP1 is present in the extracellular matrix of dentin and bone as processed NH₂- and COOH-terminal fragments. The NH₂-terminal fragment occurs as a proteoglycan, whereas the COOH-terminal fragment is highly phosphorylated. The differences in biochemical properties suggest that these fragments may have different tissue and cell distribution in association with distinct functions. In this study, we analyzed the distribution of the NH₂- and COOH-terminal fragments of DMP1 in tooth, bone, osteocytes as well as MC3T3-E1 and HEK-293 cells. Immunohistochemical analyses were performed using antibodies specific to the NH₂- or COOH-terminal region of DMP1. Clear differences in the distribution of these fragments were observed. In the teeth and bone, the NH₂-terminal fragment was primarily located in the nonmineralized predentin and cartilage of the growth plate, while the COOH-terminal fragment accumulated in the mineralized zones. In osteocytes, the NH₂-terminal fragment appeared more abundant along cell membrane and processes of osteocytes, while the COOH-terminal fragment was often found in the nuclei. This pattern of distribution in cellular compartments was further confirmed by analyses on MC3T3-E1 and HEK-293 cells transfected with a construct containing DMP1 cDNA. In these cell lines, the COOH-terminal fragment accumulated in cell nuclei, while the NH₂-terminal fragment was in the cytosol. The different distribution of DMP1 fragments indicates that these DMP1 variants must perform distinct functions.

Dentin matrix protein 1 (DMP1) is present in the extracellular matrix (ECM) of dentin and bone as processed NH₂- and COOH-terminal fragments, resulting from proteolytic cleavage at the NH₂ termini of 4 aspartic acid residues during rat DMP1 processing. One cleavage site residue, Asp¹⁸¹ (corresponding to Asp¹⁹⁷ of mouse DMP1), and its flanking region are highly conserved across species. We speculate that cleavage at the NH₂ terminus of Asp¹⁹⁷ of mouse DMP1 represents an initial, first-step scission in the whole cascade of proteolytic processing. To test if Asp¹⁹⁷ is critical for initiating the proteolytic processing of mouse DMP1, we substituted Asp¹⁹⁷ with Ala¹⁹⁷ by mutating the corresponding nucleotides of mouse cDNA that encode this amino acid residue. This mutant DMP1 cDNA was cloned into a pcDNA3.1 vector. Data from transfection experiments indicated that this single substitution blocked the proteolytic processing of mouse DMP1 in HEK-293 cells, indicating that cleavage at the NH₂ terminus of Asp¹⁹⁷ is essential for exposing other cleavage sites for the conversion of DMP1 to its fragments. The NH₂-terminal fragment of DMP1 occurs as a proteoglycan form (DMP1-PG) that contains a glycosaminoglycan (GAG) chain. Previously, we showed that a GAG chain is linked to Ser⁷⁴ in rat DMP1 (Ser⁸⁹ in mouse DMP1). To confirm that mouse DMP1-PG possesses a single GAG chain attached to Ser⁸⁹, we substituted Ser⁸⁹ by Gly⁸⁹. Data from transfection analysis indicated that this substitution completely prevented formation of the GAG-containing form, confirming that DMP1-PG contains a single GAG chain attached to Ser⁸⁹ in mouse DMP1.