Ohnishi T, Arakaki N, Nakamura O, Hirono S, Daikuhara Y
Department of Biochemistry, Kagoshima University Dental School, Japan.
J Biol Chem. 1991 Aug 5;266(22):14636-45.
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.
以大鼠部分纯化的非胶原蛋白骨基质蛋白为抗原，制备了一种针对不含磷的矿化组织特异性唾液酸蛋白的单克隆抗体。然后，以该单克隆抗体为标记，通过高效液相色谱法从大鼠下颌骨中纯化该唾液酸蛋白。分子量为59,000的唾液酸蛋白（59-kDa骨唾液酸蛋白（BSP））含有1.4%的唾液酸，但未检测到磷。用该抗体进行的免疫组织化学研究表明，该蛋白对骨和牙本质等矿化组织具有特异性，存在于成骨细胞、骨细胞和骨基质中。其他软组织，如软骨、肝脏、肾脏和骨膜，均未染色。然而，蛋白质印迹分析表明血浆中含有免疫反应性的59-kDa BSP。59-kDa BSP的定量氨基酸组成与人α2-HS糖蛋白（α2-HSG）相似（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）以及大鼠64-kDa蛋白（Franzén, A., and Heinegård, D. (1985) in The Chemistry and Biology of Mineralized Tissues (Butler, W.T., ed), p. 132, EBSCO Media, Birmingham, AL）。对59-kDa BSP的氨基末端区域和四个肽段进行的氨基酸序列分析表明，约50%的氨基酸与人α2-HSG的氨基酸同源，已知α2-HSG由肝脏合成，在血液中运输并整合到钙化组织中。但是，当用放射性亮氨酸孵育新生大鼠颅骨、富含成骨细胞的原代培养物和成年大鼠肝细胞时，通过荧光显影在它们的条件培养基中检测到了免疫反应性的59-kDa BSP。包括氨基酸序列在内的几个特征表明，59-kDa BSP可能是人类α2-HSG的大鼠对应物。然而，大鼠59-kDa BSP是一种单一肽段，由成骨细胞和肝细胞共同合成，而人类α2-HSG已知是一种异二聚体，由肝脏合成。
