Key Site Mutations Block Cleavage and Glycosylation of Murine DMP1

Objectives: In both bone and dentin, DMP1 is processed into N-terminal and C-terminal fragments at four cleavage sites and the N-terminal fragment contains a glycosaminoglycan (GAG) chain, designated DMP1-PG. To begin to determine if proteolytic processing and glycosylation are essential for the function of DMP1, we mutated one cleavage site (Asp²¹³ to Ala, designated the D213A mutation) and the GAG chain attachment site (Ser¹⁰⁵ changed to Gly, referred to as S105G mutation). Methods: Mutations were performed on normal control murine DMP1 cDNA. The cDNA fragments with desired mutations were subcloned into the pcDNA3 expression vector containing a CMV promoter. Expression constructs with mutated DMP1 cDNA or with control DMP1 cDNA were transfected into HEK-293 cells. DMP1 and its secreted fragments were analyzed by SDS-PAGE, Stains-All staining and Western immunoblotting using antibodies specific to the N-terminal and C-terminal regions of mouse DMP1. Results: In the medium of HEK-293 cells transfected with the pcDNA3 vector containing the control murine DMP1 cDNA, full-length DMP1 as well as N-terminal and C-terminal fragments were detected. In the cells transfected with the vector containing the D213A mutation, little detectable DMP1 N-terminal and C-terminal fragments were observed whereas increased secretion of intact, full-length DMP1 was clearly evident. In the cells transfected with the vector containing S105G mutation, DMP1-PG was completely absent. Conclusions: 1).The finding that mutation of Asp²¹³ almost completely blocks the proteolytic processing of mouse DMP1 suggests that this residue is the key site for initiation of the cleavage cascade for DMP1 processing. 2). The observation that the S105G mutation completely prevents the formation of the GAG-containing form of DMP1 further confirms our previous observations that murine DMP1 contains a single GAG chain at S¹⁰⁵. Characterization of these processing and glycosylation sites will help determine DMP1 functions. Supported by NIH grant DE005092 (CQ).