Blocking of proteolytic processing and deletion of glycosaminoglycan side chain of mouse DMP1 by substituting critical amino acid residues.
Dentin matrix protein 1 (DMP1) is present in the extracellular matrix (ECM) of dentin and bone as processed NH(2)- and COOH-terminal fragments, resulting from proteolytic cleavage at the NH(2) termini of 4 aspartic acid residues during rat DMP1 processing. One cleavage site residue, Asp(181) (corresponding to Asp(197) of mouse DMP1), and its flanking region are highly conserved across species. We speculate that cleavage at the NH(2) terminus of Asp(197) of mouse DMP1 represents an initial, first-step scission in the whole cascade of proteolytic processing. To test if Asp(197) is critical for initiating the proteolytic processing of mouse DMP1, we substituted Asp(197) with Ala(197) by mutating the corresponding nucleotides of mouse cDNA that encode this amino acid residue. This mutant DMP1 cDNA was cloned into a pcDNA3.1 vector. Data from transfection experiments indicated that this single substitution blocked the proteolytic processing of mouse DMP1 in HEK-293 cells, indicating that cleavage at the NH(2) terminus of Asp(197) is essential for exposing other cleavage sites for the conversion of DMP1 to its fragments. The NH(2)-terminal fragment of DMP1 occurs as a proteoglycan form (DMP1-PG) that contains a glycosaminoglycan (GAG) chain. Previously, we showed that a GAG chain is linked to Ser(74) in rat DMP1 (Ser(89) in mouse DMP1). To confirm that mouse DMP1-PG possesses a single GAG chain attached to Ser(89), we substituted Ser(89) by Gly(89). Data from transfection analysis indicated that this substitution completely prevented formation of the GAG-containing form, confirming that DMP1-PG contains a single GAG chain attached to Ser(89) in mouse DMP1.
Peng
Tao
T
Department of Biomedical Science, Baylor College of Dentistry, Texas A&M University System Health Science Center, Dallas, Tex. 75246, USA.
Huang
Bingzhen
B
Sun
Yao
Y
Lu
Yongbo
Y
Bonewald
Lynda
L
Chen
Shuo
S
Butler
William T
WT
Feng
Jerry Q
JQ
D'Souza
Rena N
RN
Qin
Chunlin
C
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D051379:Mice; D011499:Protein Processing, Post-Translational; D017384:Sequence Deletion
C513308:Dmp1 protein, mouse; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans