Design and application of new fluoroescent substrate against membrane-bound processing enzyme

点击翻译按钮获取中文摘要

RANKL (receptor activator of NF-KB ligand), an essential factor of osteoclastogenesis, exists as a membrane-bound and soluble form. Our aim was to identify its processing enzyme.First, to establish soluble RANKL expression system, ST2 cells, preosteoclast like cells, were treated by vitamine D3 (Vit D3) and dexamethasone (Dex). In general, RANKL is not produced in ST2 cells. But when ST2 cells were treated with Vit D3 and Dex, soluble form (MW ; about 30 kDa) was secreted into culture medium. Identification of soluble form was carried out by LRP (ligand-receptor precipitation) method. LRP is a new Western blot analysis that can specifically concentrate the target protein by using specific binding between RANKL and OPG (osteoprotegerin).Next, N-terminal ammo acid sequences of soluble RANKL were decided. The cleavage of membrane-bound to soluble form occurred between Arg (138) and Phe (139), and its cleavage was inhibited by KB8301, a metalloproteinase inhibitor. Currently, it is said that the candidate of processing enzyme is an ADAM family protein. Among them, mRNA expression of TACE (ADAM 17) and Kuzbanian (ADAM 10) were confirmed by RT-PCR.To examine the role of glycolipids in osteoclast formation, glycosylceramide synthase inhibitor (D-PDMP) was added to culture medium. Although the differentiation of preosteoclast to osteoclast was completely blocked by addition of D-PDMP, exogenous lactosylceramid (LacCer) significantly recovered the osteoclast formation and the phosphorylation of NF-kB and ERK inhibited by ^D PDMP. These results suggest that LacCer is important for the differentiation of osteoclast.