A Chondroitin Sulfate Chain Attached to the Bone Dentin Matrix Protein 1 NH2-Terminal Fragment

Abstract

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...

Identification of Full-Length Dentin Matrix Protein 1 in Dentin and Bone

“…This polyclonal antibody recognizes the COOH-terminal (57 kDa) fragment of DMP1 in both Western immunoblotting and immunohistochemical analyses; for the latter application, the immunoreaction of this polyclonal antibody is not as strong as that of the monoclonal antibody anti-DMP1-C-8G10.3 (see later). Both types of antibodies have been thoroughly characterized and previously used in Western immunoblotting for detection of the NH 2 -terminal or COOHterminal fragment of DMP1, respectively [13,19]. Western immunoblotting was performed as previously described [18].…”

“…For Western immunoblotting, two types of anti-DMP1 antibodies (Table 1) were used. One was a monoclonal antibody generated using the NH 2 -terminal fragment of rat bone DMP1 as the antigen [13]. This monoclonal antibody, designated anti-DMP1-N-9B6.3, reacts specifically with the NH 2 -terminal fragment of DMP1 in Western immunoblotting but shows a very weak reaction to DMP1 in immunohistochemical analysis.…”

“…In searching for naturally occurring DMP1, we discovered that the extracellular matrix (ECM) of bone and dentin contains fragments originating from intact DMP1, namely, a 37-kDa fragment from the NH 2 -terminal region and a 57-kDa fragment from the COOH-terminal region of the DMP1 amino acid sequence [12]. More recently, we discovered that the NH 2 -terminal fragment of DMP1 in the ECM of bone and dentin also occurs as a proteoglycan [13]. The proteoglycan variant, referred to as DMP1-PG, possesses a single glycosaminoglycan side chain linked to the core protein via Ser 74 in the rat DMP1 amino acid sequence.…”

“…antibody has been described previously [13] b This polyclonal antibody was used to detect the NH 2 -terminal fragment of DMP1 by immunofluorescence (primary utilization) and Western immunoblotting (secondary utilization) in this study. This antibody was recently generated in our laboratory (see "Materials and Methods") c This polyclonal antibody was used to detect the COOH-terminal fragment of DMP1 by Western immunoblotting analysis in this study.…”

The NH2-terminal and COOH-terminal Fragments of Dentin Matrix Protein 1 (DMP1) Localize Differently in the Compartments of Dentin and Growth Plate of Bone

“…However, the ECM of both tissues does contain significant amounts of processed NH 2 -terminal and COOHterminal DMP1 fragments (Qin et al 2003). Recently, a proteoglycan form of the NH 2 -terminal fragment, designated as "DMP1-PG," was discovered in bone and dentin ECM (Qin et al 2006). Biochemical analysis showed that the COOH-terminal fragment of rat DMP1 contains 41 phosphates, whereas the NH 2 -terminal fragment has 12 phosphates.…”