title: Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites.
abstract: Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH2-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe173 and Ser180 and were thus COOH termini of 37K fragments. Two peptides were from the NH2 termini of 57K fragments, starting at Asp218 and Asp222. These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe173-Asp174, Ser180-Asp181, Ser217-Asp218, and Gln221-Asp222, forming eight fragments. The uniformity of cleavages at the NH2-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.
authors.affiliation: Department of Basic Sciences, The University of Texas-Houston Health Science Center, Dental Branch, Houston, Texas 77030, USA. Chunlin.Qin@uth.tmc.edu
authors.forename: Chunlin
authors.initials: C
authors.lastname: Qin
authors.forename: Jan C
authors.initials: JC
authors.lastname: Brunn
authors.forename: Richard G
authors.initials: RG
authors.lastname: Cook
authors.forename: Ralph S
authors.initials: RS
authors.lastname: Orkiszewski
authors.forename: James P
authors.initials: JP
authors.lastname: Malone
authors.forename: Arthur
authors.initials: A
authors.lastname: Veis
authors.forename: William T
authors.initials: WT
authors.lastname: Butler
mesh_terms: D000469:Alkaline Phosphatase / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001842:Bone and Bones / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; C081635:Dmp1 protein, rat; D016326:Extracellular Matrix Proteins; D010455:Peptides; D010750:Phosphoproteins; D020033:Protein Isoforms; D014157:Transcription Factors; D000469:Alkaline Phosphatase; D014357:Trypsin