Abstract
A monoclonal antibody was raised against a mineralized tissue-specific sialoprotein containing no phosphorus using partially purified noncollagenous bone matrix proteins from rats as antigen. Then the sialoprotein was purified by high performance liquid chromatography from rat mandibulae using the monoclonal antibody as a marker. The sialoprotein (59-kDa bone sialoprotein (BSP)) with a molecular weight of 59,000 contained 1.4% sialic acid but no detectable phosphorus. Immunohistochemical studies with the antibody showed that the protein was specific to mineralized tissues such as bone and dentin, and present in osteoblasts, osteocytes, and bone matrix. No other soft tissues, such as the cartilage, liver, kidney, and periosteum, were stained. However, Western blot analysis showed that plasma contained immunoreactive 59-kDa BSP. The quantitative amino acid composition of 59-kDa BSP resembled that of human alpha 2-HS glycoprotein (alpha 2-HSG) (Lee, C.-C., Bowman, B.H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407; Kellermann, J., Haupt, H., Auerswald, E.-A., and Muller-Esterl, W. (1989) J. Biol. Chem. 264, 14121-14128) and rat 64-kDa protein (Franzén, A., and Heinegård, D. (1985) in The Chemistry and Biology of Mineralized Tissues (Butler, W.T., ed), p. 132, EBSCO Media, Birmingham, AL). Amino acid sequence analyses of the amino-terminal region and four peptide fragments of 59-kDa BSP revealed that about 50% of the amino acids were homologous with those of human alpha 2-HSG, which is known to be synthesized by the liver, transported in the bloodstream, and incorporated into calcified tissues. But when newborn rat calvaria, primary cultures of osteoblast-rich cells, and adult rat hepatocytes were incubated with radioactive leucine, immunoreactive 59-kDa BSP was detected in their conditioned medium by fluorography. Several characteristics, including the amino acid sequence, suggest that 59-kDa BSP may be the rat counterpart of human alpha 2-HSG. However, rat 59-kDa BSP is a single peptide and synthesized by both osteoblasts and hepatocytes, whereas human alpha 2-HSG is known to be a heterodimer and to be synthesized by the liver.
Highlights
Purification, Characterization, andStudies on Biosynthesis of a 59-kDa Bone Sialic Acid-containing Protein (BSP) from Rat Mandible Using a Monoclonal Antibody
Several charac- An advantage in use of monoclonal antibody directed against teristics, including the amino acid sequence, suggest a certain protein is that the antigen need not necessarily be that 59-kDa BSmPay bethe rat counterpart of human homogeneous because the hybridomas can be screened in a2-HSG
By an Enzyme-linked Immunosorbent Assay (ELISA) for 59-kDa BSP, fractions in the peak I1 shown in Fig. 1contained asmall amount of 59-kDa BSP, but more than 95% of this sialoprotein was eluted in peak I
Summary
Of 0-1.0 M NaCl in buffer A, and theabsorbance of the eluate at 280 nm was monitored. For measurement of the rateof total protein synthesis, conditioned medium obtained as described above wastreated with trichloroacetic acid at a final concentration of 10%.The samples stood for 30 min at 0 "C, and insoluble material was collected by centrifugation at 8,000 X g for 5 min at 4 "C and washed in succession twice with 0.75 ml of ice-cold 10%trichloroacetic acid containing 10 p M I-leucine, twice with 0.75 ml of HC1-acetone (0.5 ml of 12 N HCI in 100 ml of acetone), and twice with 0.75 ml of acetone It was dried under reduced pressure, and the residue was solubilized in 0.3 mlof 4 N NaOH and neutralized with 4 N HCl. The radioactivity of the solution was measured in aliquid scintillation counter(model LSC 903S, Aloka Corp., Tokyo, Japan). Sialic acid was assayed by the method described by Jourdian et al [38], and concentrations of NaCl were determined by measurement of conductivity
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