Dentin sialoprotein isoforms: detection and characterization of a high molecular weight dentin sialoprotein.
Dentin sialoprotein (DSP) is a glycoprotein accounting for 5-8% of the dentin non-collagenous proteins. The cDNA sequence predicts that rat DSP has 13 potential casein kinase phosphorylation sites and six potential N-linked glycosylation sites. However, its total phosphorylation level, as well as the nature and locations of the carbohydrate moieties, are unknown. Our findings in the present study show that rat DSP has 6.2 phosphates per molecule and that the majority of carbohydrates are attached to the protein through N-linked glycosylations. During our separation of dentin non-collagenous proteins with ion-exchange chromatography, we observed high molecular weight components eluting late in the salt gradient that were recognized by anti-DSP antibodies. We have purified these high molecular weight components using a monoclonal anti-DSP antibody affinity column. Data from amino acid analysis, phosphate level measurements and Edman degradation of tryptic peptides unequivocally proved that the very acidic, high molecular weight components are isoforms of DSP (designated HMW-DSP). Deglycosylation analysis indicates that the slower migration rate of HMW-DSP on SDS-PAGE results from its higher level of carbohydrate modifications.
Qin
Chunlin
C
Department of Basic Sciences, The University of Texas-Houston Health Science Center, Dental Branch, 6516 M.D. Anderson Boulevard, DBB, Room 4.133, Houston, TX 77030, USA. Chunlin.Qin@uth.tmc.edu
Brunn
Jan C
JC
Baba
Otto
O
Wygant
James N
JN
McIntyre
Bradley W
BW
Butler
William T
WT
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002241:Carbohydrates / Q000032:analysis; D003804:Dentin / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000032:analysis; D006031:Glycosylation; D008970:Molecular Weight; D010710:Phosphates / Q000032:analysis; D010750:Phosphoproteins; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry; D011498:Protein Precursors; D051381:Rats; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000302:isolation & purification*; D000099311:Dentin Sialophosphoprotein
D002241:Carbohydrates; D016326:Extracellular Matrix Proteins; D010710:Phosphates; D010750:Phosphoproteins; D020033:Protein Isoforms; D011498:Protein Precursors; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein