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

以部分纯化的大鼠非胶原骨基质蛋白为抗原，制备了一种抗矿化组织特异性唾液酸蛋白的单抗。以该单抗为标记物，采用高效液相色谱法从大鼠下颌骨中分离纯化唾液酸蛋白。该唾液酸蛋白(59 kDa骨涎蛋白；59 kDa-BSP)分子量为59,000，含1.4%唾液酸，不含磷。抗体的免疫化学研究表明，该蛋白特异性地存在于骨和牙本质等矿化组织中，存在于成骨细胞、骨细胞和骨基质中。软骨、肝、肾、骨膜等软组织均未见染色。而Western印迹分析表明，血浆中含有免疫活性的59 kDa-BSP。59 kDa-BSP的氨基酸组成与人α_2-HS糖蛋白(A2HSG)相似。氨基酸序列分析表明，59 kDa-BSP的氨基末端和四个肽片段与人A2HSG的氨基酸序列有约50%的同源性。接下来，我们研究了59 kDa-BSP的生物合成。新生大鼠颅骨与放射性亮氨酸孵育后，用荧光法在其条件培养液中检测到-59 kDa-BSP。原代培养的成骨细胞和成年大鼠肝细胞的条件培养液中也含有抗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能合成59 kDa的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A2HSG的大鼠对应物。然而，大鼠59 kDa-BSP是一种单肽，而人A2HSG是一种由肝脏合成、在血流中运输并结合到钙化组织中的异源二聚体。

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.