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Molecular biological research in the differentiation of osteoclast

破骨细胞分化的分子生物学研究

基本信息

批准号：
08457508
负责人：
AKAMINE Akifumi
金额：
$ 3.84万
依托单位：
KYUSHU UNIVERSTIY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至无数据
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457508/
关键词：
aspartic proteinase cathepsin D cathepsin E monomeric mutant bone resorption osteoblast immunocytochemistry

项目摘要

1. Biochemical properties of the monomeric mutant of human cathepsin E expressed in chinese hamster ovary cellsTo understand the physiological siginificance of the dimer formation, the monomeric mutant of human Cathepsin E (CE) was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Immunolocalization of the mutant protein at both the light and electron microscopic levels revealed the monomeric CE to be associated predominantly with the endoplasmic reticulum and the non-lysosomal endocytic organelles. The monomeric protein was generated primarily as the 46-kDa pro-CE with a high-mannosetype oligosaccharide chain in the cells. In contrast, the dimeric pro-CE was scarcely activated by treatment at pH 7.The monomeric form was more unstable to pH7 and temperature changes than these dimeric forms. These results indicate that the dimerization of the CE is not necessarily required for proper folding to express activity, correct intracellular localization and … More carbohydrate modification, but that it may be essential to structurally stabilize the molecule in vivo.2. Immunocytochemical demonstration of cathepsin D in the osteoblastic cells modulating bone resorptionImmunocytochemical localization of cathepsin D,a lysosomal aspartic proteinase, was investigated using immunogold methods. When the process in the osteoblastic cells partially contacted with the preosteoclasts, cathepsin D-positive particles were found within lysosome-like structures (lysosomes/endosomes) in the process of whole cytoplasm which is located close to the preosteclasts. However, in the case of active osteoclasts, cathepsin D-labeling lysosome-like structures in the osteoblastic cells were scattered within the cytoplasm.The positive structures were not concentrated within the process. This localization pattern was clearly different from that of the osteoblastic cells located in contact with preosteoclasts.These results suggest that cathepsin D is involved in the proceeding of osteoclastic bone resorption through the interaction between osteoblastic cells and osteoclasts. Less

1.中国仓鼠卵巢细胞表达的人组织蛋白酶E单体突变体的生化特性为了解二聚体形成的生理意义，通过定点诱变构建人组织蛋白酶E（CE）单体突变体并在中国仓鼠卵巢细胞中表达。突变蛋白在光和电子显微镜水平上的免疫定位揭示了单体CE主要与内质网和非溶酶体内吞细胞器相关。单体蛋白主要以 46-kDa pro-CE 的形式在细胞中产生，具有高甘露糖型寡糖链。相比之下，二聚体 pro-CE 在 pH 7 下几乎不被激活。与这些二聚体形式相比，单体形式对 pH7 和温度变化更不稳定。这些结果表明，CE 的二聚化不一定是正确折叠以表达活性、正确的细胞内定位和碳水化合物修饰所必需的，但它可能对于体内分子的结构稳定至关重要。2。组织蛋白酶 D 在成骨细胞中调节骨吸收的免疫细胞化学演示使用免疫金方法研究了组织蛋白酶 D（一种溶酶体天冬氨酸蛋白酶）的免疫细胞化学定位。当成骨细胞内的突起部分与前破骨细胞接触时，在靠近前破骨细胞的整个细胞质突起中的溶酶体样结构（溶酶体/内体）内发现组织蛋白酶D阳性颗粒。然而，在活性破骨细胞的情况下，成骨细胞中组织蛋白酶D标记的溶酶体样结构分散在细胞质内。阳性结构并未集中在过程中。这种定位模式明显不同于与前破骨细胞接触的成骨细胞的定位模式。这些结果表明组织蛋白酶D通过成骨细胞和破骨细胞之间的相互作用参与破骨细胞骨吸收的进行。较少的