Analysis of osteoclast-specificmembrane-bound proteins that are expressed during osteoclast differentiation

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It is postulated that osteoclast-specific functional proteins are expressed during the process of osteoclastic differentiation. Based on this hypothesis, we attempted the identification of an antigen (5H-antigen) for an osteoclast-specific antibody (Mab-5H) that had been established in our laboratory. Using a experimental system of osteoclastic cell formation in culture of rabbit unfractionated bone cells, we found that the 5H-antigen was expressed in the osteoclast percursors in parallel with the expression of tartrate-resistant acid phosphatase (TRAP) whichis a marker enzyme of osteoclast-diffeerntiation. When Mab-5H simultaneously with parathyroid hormone (PTH) was added into the cultures, the formation and the bone-resorbing activity of osteoclastic cells induced by PTH were abolished by the Mab-5H.These results suggest that the 5H-anitgen specifically produced by osteoclast precursors is involved in the differentiation and function of osteoclasts. In addition, 5H-antigen had two d … More ifferent forms in the osteoclasts, e.g., one was a cytosolic form and the other was a plasma membrane-bound form. Thus, we tried to purify these two distinct forms. However, it was difficult to purify the antigen from osteoclast extracts due to the small number of osteoclasts that we could harvest from the bones. Then, we sought other sources beside osteoclasts to purify the 5H-antigens, and found that intestine also expressed the 5H-antigens enough to purify them. The purified 5H-antigen in a membrane-bound form was a highly-glycosylated protein with molecular mass of 65 kDa in SDS-PAGE analysis, and was very unstable. The cytosolic antigen was also a glycoprotein with molecular mass of 600 kDa under a neutral condition and of 65 kDa in SDS-PAGE,and much more atabel the membrane-bound form. Then we sought the amino acid sequence of the cytosolic 5H-antigen. However, the amino acid analysis was unsuccessful because of a blockage of N-terminus of the antigen. We also tried to analyze the amino acid sequence of peptide fragments of the antigen generated by the limited hydrolysis, but we could not obtain the enough amount of the fragment ot be analyzed. In this study, we found the 5H-antigen was predominantly expressed in osteoclast precursors and playd an important role in osteoclast differentiation and function. However, we could not identify the 5H-antigens. Less