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Studies on Relation Between Calcification of Tissues and Biosynthesis of a 59 kDa Sialoprotein from Rat Mandible, Using a Monoclinal Antibody.

使用单斜抗体研究组织钙化与大鼠下颌骨 59 kDa 唾液酸蛋白生物合成之间的关系。

基本信息

批准号：
01480435
负责人：
DAIKUHARA Yasushi
金额：
$ 4.1万
依托单位：
Kagoshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

A monoclonal antibody was raised against a mineralized tissue-specific sialoprotein containing no phosphorus using partially purified non-collagenous bone matrix proteins from rats as antigen. Then the sialoprotein was purified by high performance liquid chromatography from rat mandibulae using the monoclinal antibody as a marker. The sialoprotein (59kDa bone sialoprotein ; 59kDa-BSP) with a molecular weight of 59,000 containging 1.4% sialic acid but no detectable phosphorus. Immunochemical 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 59kDa-BSP. The quantitative amino acid composition of 59kDa-BSP resembled that of human alpha_2-HS glycoprotein (A2HSG). Amino acid sequence analyses of the amino-terminal region and four peptide fragments of 59kDa-BSP revealed that about 50% of the amino acids were homologous with those of human A2HSG. We next examined biosynthesis of 59kDa-BSP. When newborn rat calvaria were incubated with radioactive leucine, immunoreactive-59kDa-BSP was detected in their conditioned medium by fluorography. The conditioned medium of primary cultures of osteoblast-rich cells and abult rat hepatocytes also contained radioactive material immunoprecipitated with anti-59kDa-BSP. These results indicate that both hepatocytes and osteoblasts synthesize 59kDa-BSP. Several characteristics, including the amino acid sequence, suggest that 59kDa-BSP may be the rat counterpart of human A2HSG. However, rat 59kDa-BSP is a single peptide, whereas human A2HSG is known to be a heterodimer which is synthesized by the liver, transported in the blood stream and incorporated into calcified tissues.

用部分纯化的大鼠非胶原性骨基质蛋白作为抗原，制备了一种抗矿化组织特异性唾液酸蛋白的单克隆抗体。以单克隆抗体为标记，采用高效液相色谱法从大鼠下颌骨中纯化唾液酸蛋白。唾液蛋白（59 kDa骨唾液蛋白; 59 kDa-BSP），分子量为59，000，含1.4%唾液酸，但未检测到磷。用该抗体进行的免疫化学研究表明，该蛋白对矿化组织如骨和牙本质具有特异性，并存在于成骨细胞、骨细胞和骨基质中。其他软组织如软骨、肝、肾和骨膜均未被染色。Western blot分析显示血浆中含有59 kDa-BSP。59 kDa-BSP的氨基酸组成与人α_2-HS糖蛋白（A2 HSG）相似。氨基酸序列分析表明，59 kDa-BSP与人A2 HSG的氨基酸同源性约为50%。我们接下来研究了59 kDa-BSP的生物合成。新生大鼠颅骨与放射性亮氨酸孵育后，荧光显色法检测到条件培养液中有免疫反应性~ 59 kDa-BSP。富含成骨细胞的原代培养物和成年大鼠肝细胞的条件培养基也含有用抗59 kDa-BSP免疫沉淀的放射性物质。这些结果表明，肝细胞和成骨细胞都合成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A2 HSG的大鼠对应物。然而，大鼠59 kDa-BSP是单一肽，而已知人A2 HSG是异源二聚体，其由肝脏合成，在血流中转运并掺入钙化组织中。