Morita Yoshikazu, Matsuyama Hiroaki, Serizawa Atsushi, Takeya Tatsuo, Kawakami Hiroshi
Technology and Research Institute, Snow Brand Milk Products Co., Ltd., 1-1-2 Minamidai, Kawagoe, Saitama 350-1165, Japan.
Bone. 2008 Feb;42(2):380-7. doi: 10.1016/j.bone.2007.10.012. Epub 2007 Dec 4.
We identified, for the first time, the factor responsible for inhibiting osteoclast-mediated bone resorption in the basic protein fraction of bovine milk (milk basic protein, MBP). The protein was purified by a combination of ion and gel column chromatography from MBP, based on its activity to prevent unfractionated rabbit bone cells from forming pits on dentine slices. It was found to have a molecular weight of 15 kDa on SDS-PAGE, and the sequence of the N-terminal 25 amino acid residues was identical to that of bovine angiogenin. The purified bovine angiogenin inhibited the pit-forming activity of both unfractionated bone cells and purified osteoclasts in a dose-dependent manner, and the inhibitory activity was markedly suppressed by treatment with anti-bovine angiogenin antibody. The inhibitory activity was confirmed in mice both in vitro and in vivo. Treatment of osteoclasts with bovine angiogenin resulted in an impairment of the formation F-actin ring and a reduction in the mRNA levels of TRAP and cathepsin K, both known to be essential for the bone resorption activity of osteoclasts. These results suggest that bovine angiogenin is the substance mainly responsible for the inhibitory effect of bovine milk on osteoclast-mediated bone resorption, and that it exerts its activity by acting directly on the osteoclasts.
我们首次在牛乳的碱性蛋白组分（乳碱性蛋白，MBP）中鉴定出负责抑制破骨细胞介导的骨吸收的因子。基于其防止未分级的兔骨细胞在牙本质切片上形成凹坑的活性，通过离子交换和凝胶柱色谱相结合的方法从MBP中纯化该蛋白。在SDS-PAGE上发现其分子量为15 kDa，并且N端25个氨基酸残基的序列与牛血管生成素的序列相同。纯化的牛血管生成素以剂量依赖性方式抑制未分级骨细胞和纯化破骨细胞的凹坑形成活性，并且用抗牛血管生成素抗体处理可显著抑制该抑制活性。在小鼠体内和体外均证实了该抑制活性。用牛血管生成素处理破骨细胞导致F-肌动蛋白环形成受损以及抗酒石酸酸性磷酸酶（TRAP）和组织蛋白酶K的mRNA水平降低，这两者均已知对破骨细胞的骨吸收活性至关重要。这些结果表明，牛血管生成素是牛乳对破骨细胞介导的骨吸收产生抑制作用的主要物质，并且它通过直接作用于破骨细胞发挥其活性。
