Dentin glycoprotein: the protein in the middle of the dentin sialophosphoprotein chimera

• Published in: The Journal of biological chemistry 2005-04, Vol.280 (17), p.17472-17479
• Main Authors: Yamakoshi, Yasuo, Hu, Jan C-C, Fukae, Makoto, Zhang, Hengmin, Simmer, James P
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - chemistry; Amino Acid Sequence; Amino Acids - chemistry; Animals; Asparagine - chemistry; Carbohydrate Sequence; Carbohydrates - chemistry; Chromatography; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Durapatite - chemistry; Durapatite - pharmacology; Edetic Acid - chemistry; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Glucose - chemistry; Guanidine - chemistry; Ions; Mannose - chemistry; Mass Spectrometry; Metalloendopeptidases - chemistry; Molecular Sequence Data; NF-kappa B - metabolism; Oligosaccharides - chemistry; Oxygen - metabolism; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - chemistry; Phosphoproteins - chemistry; Polysaccharides - chemistry; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Recombinant Fusion Proteins - chemistry; Sequence Homology, Amino Acid; Sialoglycoproteins - chemistry; Sialoglycoproteins - physiology; Structure-Activity Relationship; Swine; Time Factors; Tooth - embryology
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M413220200

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and is critical for proper dentin formation. DSPP is believed to be processed into only two structural/functional domains: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Here we report the isolation and characterization of a third domain of DSPP, designated dentin glycoprotein (DGP). DGP was isolated from a guanidine/EDTA extract of porcine tooth dentin by ion exchange, hydroxyapatite affinity, size exclusion, and RP-HPL chromatography. Endoproteinase lysine C digestion products of DGP were characterized by Edman sequencing and mass spectrometry. The porcine DGP backbone is the 81-amino acid segment of DSPP (Ser392 to Gly472) between the DSP and DPP domains. DGP has four phosphorylated serine residues (Ser453, Ser455, Ser457, and Ser462) and one glycosylated asparagine (Asn397). There are no other post-translational modifications. DGP is a stains-all positive protein with an apparent molecular mass on SDS-PAGE of 19 kDa, which is reduced by glycopeptidase A digestion to 16 kDa. A variety of glycans can be linked to Asn397. All are complex biantennary structures with a common N-linked pentasaccharide core (mannose3-N-acetylglucosamine2), most with a fucosyl residue on the innermost N-acetylglucosamine. The alpha1-3 and alpha1-6 arms are always galactose beta1-4 N-acetylglucosamine beta1-2 mannose, and either or both arms can be unsialidated or monosialidated. The calculated monoisotopic molecular masses of the different glycosylated forms of the DGP phosphoprotein are: unsialidated 10,523 and 10,670, monosialidated 10,815 and 10,961, and disialidated 11,106, and 11,252 Da, with the disialidated forms being the most abundant.