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

RANKL （NF-KB配体的受体激活因子）是破骨细胞形成的重要因子，以膜结合和可溶性形式存在。我们的目的是鉴定它的加工酶。首先，采用维生素D3 （Vit D3）和地塞米松（Dex）处理破骨前样细胞ST2细胞，建立可溶性RANKL表达体系。一般来说，ST2细胞不产生RANKL。而当Vit D3和Dex作用于ST2细胞时，可溶形式（MW，约30 kDa）分泌到培养基中。用LRP（配体-受体沉淀）法鉴定其可溶性形态。LRP是一种利用RANKL与OPG （osteoprotegerin）特异性结合来特异性浓缩靶蛋白的新型Western blot分析方法。然后确定可溶性RANKL的n端氨基酸序列。Arg（138）和Phe（139）之间发生膜结合到可溶性形式的裂解，其裂解受到金属蛋白酶抑制剂KB8301的抑制。目前认为加工酶的候选物是ADAM家族蛋白。其中，通过RT-PCR检测TACE （ADAM 17）和Kuzbanian (ADAM 10) mRNA的表达。为了研究糖脂在破骨细胞形成中的作用，在培养基中加入糖基神经酰胺合成酶抑制剂（D-PDMP）。虽然D-PDMP完全阻断了破骨细胞向破骨细胞的分化，但外源性乳糖酰胺（LacCer）明显恢复了破骨细胞的形成以及被D-PDMP抑制的NF-kB和ERK的磷酸化。提示LacCer对破骨细胞的分化有重要作用。

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.