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

核因子-KB配体受体激活剂(RANKL)是破骨细胞生成的重要因子，以膜结合和可溶性形式存在。首先，为了建立可溶性RANKL表达系统，用维生素D3(VitD3)和地塞米松(Dex)处理破骨前细胞ST2细胞。一般来说，在ST2细胞中不产生RANKL。而用维生素D3和地塞米松处理ST2细胞后，可见约30 kDa的可溶性蛋白分泌到培养液中。采用LRP(配基-受体沉淀法)对可溶性蛋白进行鉴定。LRP是一种新的蛋白质印迹分析方法，它利用RANKL与骨保护素(OPG)的特异性结合来特异地浓缩目的蛋白。在Arg(138)和Phe(139)之间发生了膜结合到可溶性的切割，其切割被金属蛋白酶抑制剂KB8301抑制。目前认为加工酶的候选蛋白是一种亚当家族蛋白。其中TACE(ADAM 17)和Kuzbanian(ADAM 10)的mRNA表达经RT-PCR证实。为了检测糖脂在破骨细胞形成中的作用，在培养液中加入糖基神经酰胺合成酶抑制剂(D-PDMP)。D-PDMP可完全阻断破骨前细胞向破骨细胞的分化，但外源性乳糖神经酰胺(LacCer)可显著恢复破骨细胞的形成，并能显著恢复D-PDMP抑制的核因子-kB和ERK的磷酸化。这些结果提示LacCer对破骨细胞的分化具有重要作用。

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.