Astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp)

Abstract

Dentin sialophosphoprotein (Dspp) is critical for proper dentin biomineralization because genetic defects in DSPP cause dentin dysplasia type II and dentinogenesis imperfecta types II and III. Dspp is processed by proteases into smaller subunits; the initial cleavage releases dentin phosphoprotein (Dpp). We incubated fluorescence resonance energy transfer (FRET) peptides containing the amino acid context of the Dpp cleavage site (YEFDGKSMQGDDPN, designated Dspp-FRET) or a mutant version of that context (YEFDGKSIEGDDPN, designated mutDspp-FRET) with BMP-1, MEP1A, MEP1B, MMP-2, MMP-8, MMP-9, MT1-MMP, MT3-MMP, Klk4, MMP-20, plasmin, or porcine Dpp and characterized the peptide cleavage products. Only BMP-1, MEP1A, and MEP1B cleaved Dspp-FRET at the G–D peptide bond that releases Dpp from Dspp in vivo. We isolated Dspp proteoglycan from dentin power and incubated it with the three enzymes that cleaved Dspp-FRET at the G–D bond. In each case, the released Dpp domain was isolated, and its N-terminus was characterized by Edman degradation. BMP-1 and MEP1A both cleaved native Dspp at the correct site to generate Dpp, making both these enzymes prime candidates for the protease that cleaves Dspp in vivo. MEP1B was able to degrade Dpp when the Dpp was at sufficiently high concentration to deplete free calcium ion concentration. Immunohistochemistry of developing porcine molars demonstrated that astacins are expressed by odontoblasts, a result that is consistent with RT-PCR analyses. We conclude that during odontogenesis, astacins in the predentin matrix cleave Dspp before the DDPN sequence at the N-terminus of Dpp to release Dpp from the parent Dspp protein. © 2011 American Society for Bone and Mineral Research.

DPP and DSP are Necessary for Maintaining TGF-β1 Activity in Dentin

“…The sequential extraction of proteins from dentin powder (40 g) of molars was carried out by the use of our previous demineralization method with 0.17 HCl and 0.95% formic acid (Tsuchiya et al, 2011). The supernatant obtained from 0.5 M acetic acid/2 M NaCl solution (AN extract) was used to isolate DSP and DPP.…”

“…The AN extract (150 mg) was fractionated by IE chromatography in a Q-Sepharose Fast Flow column (1.6 x 20 cm; GE Healthcare UK Ltd, Little Chalfont, UK) by our previous method (Tsuchiya et al, 2011). The DPP and DSP in the third (ANQ3) and high-molecular-weight (HMW)-DSP in the fourth (ANQ4) fractions were dialyzed against water, lyophilized, and characterized by SDS-PAGE and ALP-HPDL system with or without the TGF-β specific inhibitor, SB431542.…”

The importance of the SIBLING family of proteins on skeletal mineralisation and bone remodelling

“…It has been suggested that the proteolytic processing of DSPP to DPP, DSP and DGP is the activating stage in the mechanism of DSPP function (Zhang et al 2001. The cleavage of DPP from DSPP is catalysed by a group of zinc metallopeptidases that includes BMP1, and it is this fragment of DSPP that contains the ASARM peptide (Tsuchiya et al 2011). Various studies have shown DPP to be important in the formation and growth of HA as it has a strong affinity to Ca 2C when bound to collagen fibrils (Boskey et al 1990, Saito et al 1997, He et al 2005.…”

The Efficiency of Dentin Sialoprotein-Phosphophoryn Processing Is Affected by Mutations Both Flanking and Distant from the Cleavage Site*

“…Mutation of certain conserved residues outside of the P 4 -P 4 Ј region also had significant effects on processing. We also found that the effects of mutations of DSP-PP 240 on cleavage by purified recombinant human BMP1, a candidate physiological processing enzyme (5,6,15), paralleled the effects on cleavage by the Sf9 cell processing activity (13) explained by the high degree of conservation between the substrate-binding clefts of the candidate Sf9 cell and human processing enzymes.…”

“…Additional studies devoted to identifying the exact DSP-PP cleavage site by Steiglitz et al (20), von Marschall and Fisher (6), and Yamakoshi and co-workers (15,21) have been summarized by Ritchie et al (13).…”