Zhang Q, Domenicucci C, Goldberg H A, Wrana J L, Sodek J
Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Ontario, Canada.
J Biol Chem. 1990 May 5;265(13):7583-9.
Demineralizing extracts of porcine bone contain two large 66-80-kDa sialoproteins and smaller 20- and 23-kDa glycoproteins with similar chemical properties. Each protein was characterized following extraction from fetal calvariae and purification under dissociative conditions using Sepharose CL-6B, followed by fast protein liquid chromatography fractionation on hydroxyapatite and Mono Q resins. Unlike the large sialoproteins, the 20- and 23-kDa glycoproteins did not contain sialic acid. Nevertheless, affinity-purified antibodies raised against the 23-kDa protein recognized both the 20-kDa protein and a 67-kDa sialoprotein on immunoblots. These antibodies also immunoprecipitated a 60-kDa [35S]methionine-labeled protein produced by cell-free synthesis of calvarial bone mRNA, indicating that the smaller proteins were derived from the 67-kDa protein. The two sialoproteins were shown by primary sequence analysis to be secreted phosphoprotein I (SPPI, osteopontin, bone sialoprotein I) and bone sialoprotein (BSP, bone sialoprotein II). The SPPI was also characterized by its susceptibility to thrombin which produced a 23-kDa fragment, similar to the glycoprotein isolated, and a 30-kDa fragment. Amino-terminal sequence analysis of the 23- and 20-kDa proteins revealed that these proteins were derived from the carboxyl-terminal half of the SPPI molecule, the proteins showing 58% identity with human and rat, and 50% identity with mouse, SPPI sequences. Both the 23- and 20-kDa proteins appeared to be generated by the activity of an endogenous trypsin-like protease that cleaves at Arg-Ser (residues 155-156) and Lys-Ala (residues 182-183) bonds. Radiolabeling studies performed in vitro showed that the 23-kDa fragment was detectable in mineralized tissue within 4 h. The fragment was phosphorylated but, unlike SPPI, was not sulfated. The rapid generation of the 23-kDa glycoprotein and its presence in different bone tissues at different developmental stages indicate that the fragmentation of SPPI is important in bone formation and remodeling.
猪骨脱矿提取物含有两种分子量为66 - 80 kDa的大型唾液酸蛋白以及分子量较小的20 kDa和23 kDa糖蛋白，它们具有相似的化学性质。从胎儿颅骨中提取并在解离条件下使用琼脂糖CL - 6B进行纯化，随后在羟基磷灰石和Mono Q树脂上进行快速蛋白质液相色谱分离，对每种蛋白质进行了表征。与大型唾液酸蛋白不同，20 kDa和23 kDa糖蛋白不含唾液酸。然而，针对23 kDa蛋白产生的亲和纯化抗体在免疫印迹中识别20 kDa蛋白和一种67 kDa唾液酸蛋白。这些抗体还免疫沉淀了由颅骨mRNA的无细胞合成产生的一种60 kDa [35S]甲硫氨酸标记的蛋白，表明较小的蛋白源自67 kDa蛋白。通过一级序列分析表明，这两种唾液酸蛋白分别是分泌性磷蛋白I（SPPI，骨桥蛋白，骨唾液酸蛋白I）和骨唾液酸蛋白（BSP，骨唾液酸蛋白II）。SPPI的特征还在于其对凝血酶敏感，凝血酶可产生一个23 kDa的片段，类似于分离出的糖蛋白，以及一个30 kDa的片段。对23 kDa和20 kDa蛋白的氨基末端序列分析表明，这些蛋白源自SPPI分子的羧基末端一半，与人和大鼠的SPPI序列有58%的同一性，与小鼠的SPPI序列有50%的同一性。23 kDa和20 kDa蛋白似乎都是由一种内源性类胰蛋白酶的活性产生的，该酶在Arg - Ser（第155 - 156位残基）和Lys - Ala（第182 - 183位残基）键处切割。体外进行的放射性标记研究表明，4小时内在矿化组织中可检测到23 kDa片段。该片段被磷酸化，但与SPPI不同，未被硫酸化。23 kDa糖蛋白的快速产生及其在不同发育阶段的不同骨组织中的存在表明，SPPI的片段化在骨形成和重塑中很重要。
