Purification, characterization, and studies on biosynthesis of a 59-kDa bone sialic acid-containing protein (BSP) from rat mandible using a monoclonal antibody. Evidence that 59-kDa BSP may be the rat counterpart of human alpha 2-HS glycoprotein and is synthesized by both hepatocytes and osteoblasts.
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.
Ohnishi
T
T
Department of Biochemistry, Kagoshima University Dental School, Japan.
Arakaki
N
N
Nakamura
O
O
Hirono
S
S
Daikuhara
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001798:Blood Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D001842:Bone and Bones / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry; D058574:Integrin-Binding Sialoprotein; D008099:Liver / Q000378:metabolism*; D008334:Mandible; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid; D012795:Sialoglycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D060749:alpha-2-HS-Glycoprotein
C556354:AHSG protein, human; D000911:Antibodies, Monoclonal; D001798:Blood Proteins; C546871:IBSP protein, human; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D012795:Sialoglycoproteins; D060749:alpha-2-HS-Glycoprotein