Isolation of new phosphorylated glycoprotein from mineralized phase of bone that exhibits limited homology to adhesive protein osteopontin.
Extracts of the mineralized phase of rat calvaria were shown to contain bone acidic glycoprotein-75, a new phosphorylated glycoprotein which co-purifies with small bone proteoglycans through anion-exchange chromatography. Final purification of each was brought about with a subsequent hydroxyapatite step. Bone acidic glycoprotein-75 is 75,000 in molecular weight with a 29.3% molar content of acidic amino acid residues, a 7.0% (w/w) content of sialic acid, and a 7.9% molar content of organic phosphate. Its N-terminal sequence was determined as Leu-Pro-Val-Ala-Arg-Tyr-Gln-Asn-Thr-Glu-Glu-Glu-Glu-. Because the size and charge density properties of bone acidic glycoprotein-75 are similar to those reported for rat bone sialoprotein II, calvarial sialoprotein II was also purified to homogeneity, and its amino acid composition and N-terminal sequence were determined. The sequence results showed an identity with the first 5 residues of human sialoprotein II and a complete lack of homology with bone acidic glycoprotein-75, which, furthermore, did not bind anti-sialoprotein II antibodies. Although the N-terminal sequence of bone acidic glycoprotein-75 appears to be unique, a 33% homology is shared with rat adhesive protein osteopontin. Affinity-purified antibodies against osteopontin were found to specifically bind to bone acidic glycoprotein-75 and to sialoprotein II upon immunoblotting, whether as purified proteins or as components of crude calvarial extracts. In summary, bone acidic glycoprotein-75 is a new phosphorylated glycoprotein from the mineralized compartment of rat calvarial tissue with a limited structural homology to osteopontin.
Gorski
J P
JP
Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri-Kansas City 64110.
Shimizu
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001842:Bone and Bones / Q000032:analysis*; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D006882:Hydroxyapatites; D008969:Molecular Sequence Data; D008970:Molecular Weight; D053495:Osteopontin; D010750:Phosphoproteins / Q000302:isolation & purification*; D011509:Proteoglycans / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid; D012795:Sialoglycoproteins
D000596:Amino Acids; D006023:Glycoproteins; D006882:Hydroxyapatites; D010750:Phosphoproteins; D011509:Proteoglycans; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin; D017886:Durapatite