Keratan sulphate proteoglycans associated with the bone formation of embryonic chick femurs

用4种单克隆抗体（MAb 2B 6、3B 3、1B 5和5D4），对15-18天鸡胚股骨中段骨膜下骨中硫酸化蛋白多糖（PG）的类型和分布进行了免疫细胞化学和生物化学研究。这些MAb分别特异性识别4-硫酸软骨素（C4-S）和硫酸皮肤素（DS）; 6-硫酸软骨素（C6-S）和未硫酸化软骨素（C 0-S）; C 0-S ;和硫酸角质素（KS）中的表位。免疫组化结果显示，在15-18天的胚胎标本中，C4-S、DS和KS的染色仅限于类骨质、骨细胞的细胞表面以及骨细胞陷窝和骨小管的壁，而C6-S和C 0-S的染色不限于类骨质和骨细胞的细胞表面。然而，在这些标本中未观察到免疫染色的分布和强度的显著差异。从新鲜的18天胚胎标本中提取骨蛋白，用3种提取程序，4 M盐酸胍（GdnCl，G-1提取物），0.4M EDTA（E-ectract），然后用GdnCl（G-2提取物），分别表征E-和G2-提取物中矿物结合和胶原基质相关的PG。E-和G2-提取物的Western印迹分析表明，软骨素酶ABC消化的PG具有约45，000的分子量（M_r），其含有主要对应于C4-S和/或DS的GAG，在矿物和基质相中不存在可检测的C6-S或C 0-S，而具有约72，000的M_r的KSPG仅存在于矿物相中。这些结果表明，鸡胚骨中含有C4-S，DS和KS链的小PG，其优先定位于类骨质，骨细胞的细胞表面，以及骨细胞陷窝和骨小管的壁。

The type and sistribution of sulphated proteoglycans (PGs) in the midshaft subperiosteal bone of 15-18-day embryonic chick femurs were studied immunocytochemically and biochemically, using four monoclonal antibodies (MAb 2B6, 3B3, 1B5, and 5D4). These MAb specifically recognize epitopes in chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS) ; chondroitin 6-sulphate (C6-S) and unsulphated chondroitin (C0-S) ; C0-S ; and keratan sulphate (KS) respectively. Immunohistochemistry showed that staining of C4-S, DS, and KS, but not of C6-S and C0-S, was limited to osteoid, the cell surface of osteocytes, and to the walls of osteocytic lacunae and bone canaliculi in 15-18-day embryonic specimens. However, no significant difference in the distribution and intensity of immunostaining was observed in these specimens. Bone proteins were extracted from fresh 18-day embryonic specimens with a three extraction procedure, 4 M guanidine HCl (GdnCl, G-1 extract), 0.4M EDTA (E-ectract), followed by GdnCl (G-2extract), to characterize mineral binding and collagenous matrix associated PGs in E-and G2-extracts respectively. Western blot analysis of E-and G2-extracts demonstrated that chondroitinase ABC-digested PGs with a molecular weight (M_r) approximately of 45,000 containing GAGs predominantly corresponding to C4-S and/or DS, with no detectable C6-S or C0-S present in the mineral and matrix phase, whereas KSPGs having an M_r of approximately 72,000 are only present in the mineral phase. These results indicate that embryonic chick bone contains small PGs having C4-S, DS, and KS chains with preferential localization to osteoid, the cell surface of osteocytes, and to the walls of osteocytic lacunac and bone canaliculi.