1996 Fiscal Year Final Research Report Summary
Molecular biological research in the differentiation of osteoclast
Grant-in-Aid for Scientific Research (B)
|Allocation Type||Single-year Grants |
|Research Institution||KYUSHU UNIVERSTIY |
AKAMINE Akifumi Kyushu Univ., Dentistry, Professor -> 九州大学, 歯学部, 教授 (00117053)
GOTO Yasuharu Kyushu Univ., Dentistry, Research Associate, 歯学部, 助手 (00170473)
HIRATA Masako Kyushu Univ., Dentistry, Research Associate, 歯学部, 助手 (10153769)
NAKAMUTA Hiroyoshi Kyushu Univ., Dentistry, Assistant Professor, 歯学部, 講師 (20117188)
|Project Period (FY)
|Keywords||aspartic proteinase / cathepsin D / cathepsin E / monomeric mutant / bone resorption / osteoblast / immunocytochemistry|
1. Biochemical properties of the monomeric mutant of human cathepsin E expressed in chinese hamster ovary cells
To 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
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 resorption
Immunocytochemical 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