Blocking of proteolytic processing and deletion of glycosaminoglycan side chain of mouse DMP1 by substituting critical amino acid residues

Tao Peng, Bingzhen Huang, Yao Sun, Yongbo Lu, Lynda Bonewald, Shuo Chen, William T. Butler, Jerry Q. Feng, Rena N. D'Souza, Chunlin Qin

Research output: Contribution to journalArticle

12 Scopus citations

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, Asp 181 (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 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 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.

Original languageEnglish (US)
Pages (from-to)192-197
Number of pages6
JournalCells Tissues Organs
Volume189
Issue number1-4
DOIs
StatePublished - Dec 1 2008

Keywords

  • Dentin matrix protein 1
  • Glycosaminoglycan
  • Mutation
  • Proteolytic processing

ASJC Scopus subject areas

  • Anatomy
  • Histology

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