Blocking of Proteolytic Processing and Deletion of Glycosaminoglycan Side Chain of Mouse DMP1 by Substituting Critical Amino Acid Residues

Abstract

Dentin matrix protein 1 (DMP1) is present in the extracellular matrix (ECM) of dentin and bone as processed NH₂- and COOH-terminal fragments, resulting from proteolytic cleavage at the NH₂ termini of 4 aspartic acid residues during rat DMP1 processing. One cleavage site residue, Asp¹⁸¹ (corresponding to Asp¹⁹⁷ of mouse DMP1), and its flanking region are highly conserved across species. We speculate that cleavage at the NH₂ terminus of Asp¹⁹⁷ of mouse DMP1 represents an initial, first-step scission in the whole cascade of proteolytic processing. To test if Asp¹⁹⁷ is critical for initiating the proteolytic processing of mouse DMP1, we substituted Asp¹⁹⁷ with Ala¹⁹⁷ 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 NH₂ terminus of Asp¹⁹⁷ is essential for exposing other cleavage sites for the conversion of DMP1 to its fragments. The NH₂-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 Ser⁷⁴ in rat DMP1 (Ser⁸⁹ in mouse DMP1). To confirm that mouse DMP1-PG possesses a single GAG chain attached to Ser⁸⁹, we substituted Ser⁸⁹ by Gly⁸⁹. 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 Ser⁸⁹ in mouse DMP1.