Nagata T, Goldberg H A, Zhang Q, Domenicucci C, Sodek J
Medical Research Council Group in Periodontal Physiology, University of Toronto, Ontario, Canada.
Matrix. 1991 Apr;11(2):86-100. doi: 10.1016/s0934-8832(11)80212-x.
To study the biosynthesis of bone proteins, fragments of fetal porcine calvariae were cultured in the presence of 50 micrograms/ml ascorbate and 10 mM beta-glycerophosphate and individual cultures labeled for either 4 h or 48 h with [35S]-methionine, Na2[35SO4], Na3[32PO4] or [14C]-glycine plus [14C]-proline. The radiolabeled proteins in tissue extracts were obtained by sequential extraction with 4 M GuHCl (G1-extract), 0.5 M EDTA (E-extract), and again with 4 M GuHCl (G2-extract) and analyzed together with the radiolabeled proteins secreted into the medium. SPP-1 (secreted phosphoprotein 1, osteopontin) was the major non-collagenous protein deposited into the bone matrix, with lesser amounts of BSP (bone sialoprotein), osteocalcin and chondroitin sulfate proteoglycans (CS-PG II and CS-PG III). SPP-1 was also the major phosphorylated protein and was recovered, together with several fragmented forms, almost entirely in the demineralizing extracts. Moreover, approximately one-half of the [35SO4] incorporated into E-extract proteins was present in SPP-1, the remainder being incorporated into PGs with smaller amounts associated with BSP. Over 65% of the [35SO4] in the proteoglycans of the demineralizing extracts was recovered in the small CS-PG III with less than 35% in CS-PG II, the bone homologue of DS-PG II (decorin). In contrast, CS-PG II was the predominant small proteoglycan in culture media and in guanidine extracts. Some sulfated BSP was also observed in guanidine extracts and small amounts appeared to bind to collagen. Radiolabeled SPARC (osteonectin), a prominent protein of fetal porcine bone, was not detected in the mineralized bone tissues but was prominent in the culture medium. These results demonstrate that following secretion, the major proteins expressed by osteoblastic cells are initially incorporated into different tissue compartments, with most of the sulfated sialoproteins and CS-PG III associating rapidly with the hydroxyapatite crystals. The initial distribution of these proteins is of importance in the evaluation of their role in bone formation and mineralization.
为研究骨蛋白的生物合成，将胎猪颅骨碎片在含有50微克/毫升抗坏血酸和10毫摩尔β-甘油磷酸的条件下培养，并用[35S]-甲硫氨酸、Na2[35SO4]、Na3[32PO4]或[14C]-甘氨酸加[14C]-脯氨酸对单个培养物分别标记4小时或48小时。组织提取物中的放射性标记蛋白通过依次用4M盐酸胍（G1提取物）、0.5M乙二胺四乙酸（E提取物）以及再次用4M盐酸胍（G2提取物）提取获得，并与分泌到培养基中的放射性标记蛋白一起进行分析。分泌性磷蛋白1（SPP-1，骨桥蛋白）是沉积到骨基质中的主要非胶原蛋白，骨唾液蛋白（BSP）、骨钙素和硫酸软骨素蛋白聚糖（CS-PG II和CS-PG III）的含量较少。SPP-1也是主要的磷酸化蛋白，与几种片段形式一起几乎完全在脱矿质提取物中回收。此外，掺入E提取物蛋白中的[35SO4]约有一半存在于SPP-1中，其余部分掺入蛋白聚糖，与BSP相关的量较少。脱矿质提取物蛋白聚糖中超过65%的[35SO4]在小的CS-PG III中回收，在CS-PG II中不到35%，CS-PG II是DS-PG II（核心蛋白聚糖）的骨同源物。相比之下，CS-PG II是培养基和胍提取物中主要的小蛋白聚糖。在胍提取物中也观察到一些硫酸化的BSP，少量似乎与胶原蛋白结合。放射性标记的骨连接蛋白（SPARC）是胎猪骨中的一种重要蛋白，在矿化骨组织中未检测到，但在培养基中很突出。这些结果表明，分泌后，成骨细胞表达的主要蛋白最初被纳入不同的组织区室，大多数硫酸化唾液蛋白和CS-PG III迅速与羟基磷灰石晶体结合。这些蛋白的初始分布对于评估它们在骨形成和矿化中的作用很重要。
