Analysis of membrane receptor of aralin, a cancer-selective cytotoxic protein from aralin elata

阿拉林是一种新型的II型核糖体失活蛋白(RIP)。它的A链具有RNA N-糖苷酶活性，可以灭活核糖体，抑制蛋白质合成，而B链是Gal及其衍生物特有的凝集素。与正常细胞相比，阿拉林优先诱导癌细胞的凋亡。为了鉴定有效的aralin受体，我们用抗aralin抗体的Far Western blotting和LC/MS对膜蛋白进行了分析，结果表明aralin受体是110 kDa的高密度脂蛋白结合蛋白(HDLBP)，它是由150 kDa的高密度脂蛋白结合蛋白(HDLBP)加工而来的，作为一种活性高密度脂蛋白受体存在于脂质筏中。110 kDa HDLBP在各种癌细胞中的表达水平均高于正常细胞。此外，我们使用miRNAs建立了110 kDa的HDLBP基因敲除的HeLa细胞。这些细胞对阿拉林的敏感性显著降低。相反，仅强制表达150 kDa的HDLBP并不能获得110 kDa的高表达细胞。意想不到的是，这些细胞对阿拉林的敏感性与对照细胞相当。因此，这些结果表明，处理后的110 kDa HDLBP是真正的受体，其在脂筏中的表达水平决定了对阿拉林的敏感性。用N-末端和C-末端标记的150-kDa HDLBP对150-kDa到110-kDa活性形式的HDLBP进行处理分析表明，N-末端区域可以被去除。目前，我们正在进行N-末端切割位点和可能的加工酶的鉴定。此外，我们正在探索HDLBP影响多种癌细胞的处理机制。

Aralin from Aralia elatais a new type II ribosome inactivating protein (RIP). Its A-chain exhibits RNA N-glycosidase activity to inactivate the ribosome and inhibit protein synthesis, while B-chain is the Gal and its derivatives-specific lectin. Aralin preferentially induces apoptosis in cancer cells compared with normal cells. To identify the potent aralin receptor, we previously analyzed the membrane proteins by far Western blotting with anti-aralin antibody, and LC/MS. The obtained data suggested that aralin receptor is the 110-kDa high density lipoprotein binding protein (HDLBP), which is processed from 150-kDa HDLBP and existing in lipid raft as an active HDL receptor. The expression levels of 110-kDa HDLBP of various cancer cells were higher than those of normal cells. Furthermore, we established 110-kDa HDLBP-knockdown HeLa cells using miRNAs. The sensitivity of these cells against aralin was robustly reduced. In contrast, 110-kDa HDLBP-over-expressing cells were not obtained by only forced expression of 150-kDa HDLBP. Expectedly, sensitivity of these cells against aralin was comparable to the control cells. Thus, these results indicate that the processed 110-kDa HDLBP is the authentic receptor and its expression level in the lipid raft determines the sensitivity toward aralin. HDLBP processing analysis from 150-kDa to 110-kDa active form using N- and C-terminal-tagged 150-kDa HDLBP indicated that the N-terminal region could be removed. Currently, we were pursuing the identification of the N-terminal cutting site and possible processing enzymes. In addition, we are exploring the processing mechanism of the HDLBP affecting variety of cancer cells.