Astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp).
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.
Tsuchiya
Shuhei
S
Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48108, USA.
Simmer
James P
JP
Hu
Jan C-C
JC
Richardson
Amelia S
AS
Yamakoshi
Fumiko
F
Yamakoshi
Yasuo
Y
D000595:Amino Acid Sequence; D000818:Animals; D003804:Dentin / Q000201:enzymology; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009804:Odontoblasts / Q000166:cytology / Q000201:enzymology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D012795:Sialoglycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D034421:Sus scrofa; D000099311:Dentin Sialophosphoprotein
D016326:Extracellular Matrix Proteins; D008666:Metalloendopeptidases; D010455:Peptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein; C071288:astacin