DSP-PP C-Terminal Conservation Is Crucial for Accurate DSP-PP Precursor Cleavage

Area of Science:

• Biochemistry
• Molecular Biology
• Biomineralization

Background:

• Dentin Sialoprotein (DSP) and Phosphophoryn (PP) are acidic proteins essential for dentin mineralization.
• These proteins are derived from a single DSP-PP precursor, which requires proteolytic processing for functional maturation.
• Obtaining sufficient quantities of DSP-PP has been challenging, necessitating recombinant protein production.

Purpose of the Study:

• To investigate the role of the highly conserved C-terminal region of DSPP in DSP-PP precursor cleavage.
• To determine the impact of specific C-terminal mutations on the proteolytic processing of DSP-PP.
• To assess the necessity of the C-terminal DD residues for cleavage by TLR1 and BMP1.

Main Methods:

• Utilized a Sf9-baculovirus expression system to produce DSP-PP₂₄₀ recombinant protein.
• Generated a DSP-PP C-terminal mutant (DD/HH-DSP-PP) by substituting terminal aspartate residues with histamines.
• Assessed cleavage of wild-type and mutant DSP-PP using endogenous Sf9 Tolloid-Related 1 (TLR1) and exogenous recombinant Bone Morphogenic Protein 1 (BMP1) in vitro.

Main Results:

• The DD/HH mutation in DSP-PP completely blocked its cleavage into DSP and PP by both TLR1 and BMP1.
• Mass spectrometric analysis previously identified the precise BMP1 cleavage site within DSP-PP (SMQG⁴⁴⁷|D⁴⁴⁸DPN).
• Mutations at the cleavage site or outside of it can affect cleavage efficiency, indicating conserved regulatory mechanisms.

Conclusions:

• The C-terminal D⁶⁸⁶D⁶⁸⁷ residues actively participate in controlling DSP-PP precursor cleavage.
• C-terminal conservation is critical for the proper processing of DSP-PP by proteases like TLR1 and BMP1.
• This finding provides insights into the molecular mechanisms regulating dentin matrix protein maturation.