Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil.
J Cell Biochem. 2011 Sep;112(9):2529-40. doi: 10.1002/jcb.23177.
Brown spider venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown spider venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying recombinant phospholipase-D, a potent hemolytic and phospholipase-D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic sphingomyelin (SM), lysophosphatidylcholine (LPC), and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant phospholipase-D treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of calcium into the cells that was detected by a calcium-sensitive fluorescent probe (Fluo-4). This calcium influx was shown to be channel-mediated rather than leak-promoted because the influx was inhibited by L-type calcium channel inhibitors but not by a T-type calcium channel blocker, sodium channel inhibitor or a specific inhibitor of calcium activated potassium channels. Finally, this inhibition of hemolysis following recombinant phospholipase-D treatment occurred in a concentration-dependent manner in the presence of L-type calcium channel blockers such as nifedipine and verapamil. The data provided herein, suggest that the brown spider venom phospholipase-D-induced hemolysis of human erythrocytes is dependent on the metabolism of membrane phospholipids, such as SM and LPC, generating bioactive products that stimulate a calcium influx into red blood cells mediated by the L-type channel.
棕色蜘蛛毒液磷脂酶-D 属于一类毒素，其特征为强效生物活性物质。这些毒素参与了许多细胞病理生理学方面，包括炎症反应、血小板聚集、内皮细胞过度激活、肾脏疾病和溶血。这些毒素引起溶血的分子机制正在研究中；文献数据表明，酶催化对于毒素触发的生物学活性是必要的。然而，磷脂酶-D 活性与人类溶血直接相关的方式尚未确定。为了评估棕色蜘蛛毒液磷脂酶-D 活性如何引起溶血，我们检查了重组磷脂酶-D 对人红细胞的影响。使用从 cDNA 毒腺文库获得的六种不同的纯化重组磷脂酶-D 分子，我们证明了溶血效应和磷脂酶-D 活性之间存在相关性。通过研究重组磷脂酶-D，一种强效溶血和磷脂酶-D 重组毒素（LiRecDT1），我们确定该毒素降解合成鞘磷脂（SM）、溶血磷脂酰胆碱（LPC）和溶血血小板激活因子。此外，我们确定该毒素降解人红细胞去污剂提取物中的磷脂以及人红细胞胞浆中的磷脂。重组磷脂酶-D 处理后合成 SM 和 LPC 的降解产物导致人红细胞溶血。这种依赖于磷脂代谢产物的溶血也依赖于钙离子浓度，因为随着培养基中钙离子剂量的增加，溶血百分比增加。重组磷脂酶-D 处理人红细胞刺激钙离子内流入细胞，这可以通过钙敏荧光探针（Fluo-4）检测到。这种钙离子内流是由通道介导的，而不是由漏出促进的，因为内流被 L 型钙通道抑制剂抑制，但不受 T 型钙通道阻滞剂、钠通道抑制剂或钙激活钾通道的特异性抑制剂抑制。最后，在存在 L 型钙通道阻滞剂（如硝苯地平和维拉帕米）的情况下，重组磷脂酶-D 处理后溶血的抑制呈浓度依赖性。本文提供的数据表明，棕色蜘蛛毒液磷脂酶-D 诱导的人红细胞溶血依赖于膜磷脂（如 SM 和 LPC）的代谢，产生生物活性产物，刺激 L 型通道介导的红细胞内钙离子流入。
