Association of bovine dentine phosphophoryn with collagen fragments.
Bovine dentine phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser(P)) residues, is synthesized by odontoblasts and believed to be involved in matrix-mediated biomineralization of dentine. Phosphophoryn was purified from bovine dentine using EDTA extraction, Ca(2+) precipitation, anion exchange and size exclusion chromatography. The purified protein migrated on SDS-PAGGE as a single band. The protein was dephosphorylated using a chelex alkaline dialysis procedure, repurified using anion exchange and size exclusion chromatography and then subjected to cleavage with trypsin. The digest was subjected to reversed-phase HPLC and analysed by Q-TOF mass spectrometry. The only non-trypsin peptides that could be identified were two collagen Type I alpha2 peptides whose sequence was determined by fragmentation analysis. The association of collagen fragments with highly purified phosphophoryn suggests that the EDTA extraction method yields BDP that is strongly bound to collagen fragments. This association now helps explain discrepancies in molecular weight and amino acid composition data for various phosphophoryn preparations compared with the same data calculated from the C-terminal extension of mouse, rat and human dentine sialophosphoprotein (DSPP) gene products. Analysis of the mutation pattern of the clinical disorder Osteogenesis Imperfecta within the region enclosed by the identified collagen fragments reveals that phosphophoryn associates with a segment of collagen that is crucial for structure and/or function.
Huq
N Laila
NL
Centre for Oral Health Science, School of Dental Science, The University of Melbourne, 711 Elizabeth Street, Melbourne, Victoria 3000, Australia.
Loganathan
Ashwini
A
Cross
Keith J
KJ
Chen
Yu-Yen
YY
Johnson
Neil I
NI
Willetts
Matthew
M
Veith
Paul D
PD
Reynolds
Eric C
EC
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002845:Chromatography / Q000379:methods; D003094:Collagen / Q000032:analysis*; D024042:Collagen Type I; D003804:Dentin / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D024042:Collagen Type I; D010750:Phosphoproteins; C017630:phosphophoryn; D003094:Collagen; D014357:Trypsin