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) 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 ...
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