Characterization of fetal porcine bone sialoproteins, secreted phosphoprotein I (SPPI, osteopontin), bone sialoprotein, and a 23-kDa glycoprotein. Demonstration that the 23-kDa glycoprotein is derived from the carboxyl terminus of SPPI.
Demineralizing extracts of porcine bone contain two large 66-80-kDa sialoproteins and smaller 20- and 23-kDa glycoproteins with similar chemical properties. Each protein was characterized following extraction from fetal calvariae and purification under dissociative conditions using Sepharose CL-6B, followed by fast protein liquid chromatography fractionation on hydroxyapatite and Mono Q resins. Unlike the large sialoproteins, the 20- and 23-kDa glycoproteins did not contain sialic acid. Nevertheless, affinity-purified antibodies raised against the 23-kDa protein recognized both the 20-kDa protein and a 67-kDa sialoprotein on immunoblots. These antibodies also immunoprecipitated a 60-kDa [35S]methionine-labeled protein produced by cell-free synthesis of calvarial bone mRNA, indicating that the smaller proteins were derived from the 67-kDa protein. The two sialoproteins were shown by primary sequence analysis to be secreted phosphoprotein I (SPPI, osteopontin, bone sialoprotein I) and bone sialoprotein (BSP, bone sialoprotein II). The SPPI was also characterized by its susceptibility to thrombin which produced a 23-kDa fragment, similar to the glycoprotein isolated, and a 30-kDa fragment. Amino-terminal sequence analysis of the 23- and 20-kDa proteins revealed that these proteins were derived from the carboxyl-terminal half of the SPPI molecule, the proteins showing 58% identity with human and rat, and 50% identity with mouse, SPPI sequences. Both the 23- and 20-kDa proteins appeared to be generated by the activity of an endogenous trypsin-like protease that cleaves at Arg-Ser (residues 155-156) and Lys-Ala (residues 182-183) bonds. Radiolabeling studies performed in vitro showed that the 23-kDa fragment was detectable in mineralized tissue within 4 h. The fragment was phosphorylated but, unlike SPPI, was not sulfated. The rapid generation of the 23-kDa glycoprotein and its presence in different bone tissues at different developmental stages indicate that the fragmentation of SPPI is important in bone formation and remodeling.
Zhang
Q
Q
Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Ontario, Canada.
Domenicucci
C
C
Goldberg
H A
HA
Wrana
J L
JL
Sodek
J
J
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D001842:Bone and Bones / Q000378:metabolism*; D002474:Cell-Free System; D002846:Chromatography, Affinity; D005333:Fetus; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D053495:Osteopontin; D010750:Phosphoproteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D012795:Sialoglycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D013552:Swine
D006023:Glycoproteins; C546871:IBSP protein, human; C546872:Ibsp protein, mouse; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D010750:Phosphoproteins; D012333:RNA, Messenger; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; C506897:Spp1 protein, rat; D053495:Osteopontin