Dentin sialophosphoprotein (DSPP) is cleaved into its two natural dentin matrix products by three isoforms of bone morphogenetic protein-1 (BMP1)

Abstract

The protease that cleaves the most abundant non-collagenous protein of dentin matrix, dentin sialophosphoprotein (DSPP), into its two final dentin matrix products, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), has not been directly identified. In this study, full-length recombinant mouse DSPP was made for the first time in furin-deficient mammalian LoVo cells and used to test the ability of three different isoforms of one candidate protease, bone morphogenetic protein-1 (BMP1) to cleave DSPP at the appropriate site. Furthermore, two reported enhancers of BMP1/mTLD activity (procollagen C-endopeptidase enhancer-1, PCPE-1, and secreted frizzled-related protein-2, sFRP2) were tested for their abilities to modulate BMP1-mediated processing of both DSPP and another SIBLING family member with a similar cleavage motif, dentin matrix protein-1 (DMP1). Three splice variants of BMP1 (classic BMP1, the full-length mTolloid (mTLD), and the shorter isoform lacking the CUB3 domain, BMP1-5) were all shown to cleave the recombinant DSPP in vitro although mTLD was relatively inefficient at processing both DSPP and DMP1. Mutation of the MQGDD peptide motif to IEGDD completely eliminated the ability of all three recombinant isoforms to process full-length recombinant DSPP in vitro thereby verifying the single predicted cleavage site. Furthermore when human bone marrow stromal cells (which naturally express furin-activated BMP1) were transduced with the adenovirus-encoding either wild-type or mutant DSPP, they were observed to fully cleave wild-type DSPP but failed to process the mutant DSPPMQΔIE during biogenesis. All three BMP1 isoforms were shown to process type I procollagen as well as DSPP and DMP1 much more efficiently in low salt buffer (≤50 mM NaCl) compared to commonly used normal saline buffers (150 mM NaCl). Neither PCPE-1 nor sFRP2 were able to enhance any of the three BMP1 isoforms in cleaving either DSPP or DMP1 under either low or normal saline conditions. Interestingly, we were unable to reproduce sFRP2’s reported ability to enhance the processing of type I procollagen by BMP1/mTLD. In summary, three isoforms of BMP1 process both DSPP and DMP1 at the MQX/DDP motif, but the identity of a protein that can enhance the cleavage of the two SIBLING proteins remains elusive.

DSPP Is Essential for Normal Development of the Dental-Craniofacial Complex

“…Immediately after its synthesis, DSPP is cleaved into DSP and DPP. BMP1, MMP2, and MMP20 have been implicated in the proteolytic process (Yamakoshi et al 2006;von Marschall and Fisher 2010). In this study, we demonstrate that the DSPP gene is essential for the formation of mineralized tissues of the dentalcraniofacial complex.…”

Sizzled Is Unique among Secreted Frizzled-related Proteins for Its Ability to Specifically Inhibit Bone Morphogenetic Protein-1 (BMP-1)/Tolloid-like Proteinases

“…In contrast, He et al (28) showed that high concentrations of sFRP-2 inhibited the cleavage of a fluorogenic peptide and of procollagen I by human BMP-1, and using a similar model, they concluded that direct injection of sFRP-2 in the infracted heart could inhibit fibrosis and improve cardiac function. Finally, a third study from von Marschall et al (29) found that sFRP-2 had no effect on the procollagen-C-proteinase activity of BMP-1.…”

Metalloproteinases in Drosophila to Humans That Are Central Players in Developmental Processes

“…Attempting to explain the apparently contradictory observations of decreased fibrosis in sFRP2-null mice (97) and decreased fibrosis upon injection of sFRP2 into rat heart with MI, He et al (98) suggested that reduced MI fibrosis in sFRP2-null mice may result from apoptotic loss of fibrogenic cells due to loss of sFRP2 anti-apoptotic activity. A third study reported no effect of sFRP2 on the in vitro pCP activity of BMP1 or mTLD (61).…”

“…Both are secreted as zymogens that are activated by B/TPs via cleavage of prodomains (59,60). Dentin matrix protein 1 (DMP1) and DSPP (dentin sialophosphoprotein), SIBLING family members, are highly acidic proteins that can be cleaved by B/TPs to produce fragments involved in initiating mineralization of hard tissues (61,62). Observations that DMP1-processing activity is decreased in cells null for BMP1, mTLD, and mTLL1 (62) and that expression of both BMP1 and DMP1 increases coincident with mineralization (63) are supportive of the physiological relevance for B/TP cleavage of DMP1.…”