Haemolytic activity of stonustoxin from stonefish (Synanceja horrida) venom: pore formation and the role of cationic amino acid residues
Haemolytic activity of stonustoxin from stonefish (Synanceja horrida) venom: pore formation and the role of cationic amino acid residues
复制标题
DOI：
10.1042/bj3250685
复制
发表时间：
1997-08-01
影响因子：
4.1
通讯作者：
Khoo, HE
中科院分区：
文献类型：
--
作者：
Chen, DS;Kini, RM;Khoo, HE
石鱼毒素（Stonustoxin，SNTX）是从石鱼（Synanceja horrida）毒液中纯化的一种双亚基蛋白毒素，其诱导强烈的溶血活性。我们通过渗透保护试验研究了这种非酶蛋白的成孔特性。SNTX诱导的溶血被足够大小的渗透保护剂完全阻止[聚（乙二醇）3000;分子直径约为10000]。3.2 nm]。较小尺寸的不带电分子，如棉子糖和聚乙二醇1000- 2000，不能防止细胞裂解。这些发现表明SNTX诱导细胞膜中亲水性孔的形成，这导致红细胞溶解。由于阳离子残基有助于显着的细胞溶解活性的其他几个成孔毒素，我们研究了带正电荷的赖氨酸和精氨酸残基的溶血活性的SNTX的作用。当赖氨酸残基的带正电荷的侧链分别在氨甲酰化或琥珀酰化后被中和或转化为带负电荷的侧链时，SNTX失去其溶血活性。用2，3-丁二酮修饰带正电荷的精氨酸残基也能抑制SNTX的溶血活性。溶血的损失与修饰的Lys或Arg残基的数量有很强的相关性。然而，CD分析表明，SNTX的构象没有显着影响这些化学修饰。此外，SNTX的溶血活性被各种带负电荷的脂质如磷脂酰丝氨酸、心磷脂和单唾液酸神经节苷脂竞争性抑制。这些结果表明，SNTX通过在细胞膜中形成孔诱导有效的溶血活性;并且阳离子残基在其细胞溶解机制中起关键作用。
Stonustoxin (SNTX) is a two-subunit protein toxin purified from the venom of the stonefish (Synanceja horrida), which induces potent haemolytic activity. We examined the pore-forming property of this non-enzymic protein by an osmotic protection assay. SNTX-induced haemolysis was completely prevented by osmotic protectants of adequate size [poly(ethylene) glycol 3000; molecular diameter approx. 3.2 nm]. Uncharged molecules of smaller size, such as raffinose and poly(ethylene) glycol 1000- 2000, failed to protect against cell lysis. These findings indicate that SNTX induces the formation of hydrophilic pores in the cell membrane, which results in the lysis of erythrocytes. Since cationic residues contribute significantly to the cytolytic activity of several other pore-forming toxins, we examined the role of positively charged lysine and arginine residues in the haemolytic activity of SNTX. SNTX lost its haemolytic activity when the positively charged side chains of lysine residues were neutralized or converted into negatively charged side chains upon carbamylation or succinylation respectively. The haemolytic activity of SNTX was also inhibited by the modification of positively charged arginine residues using 2,3-butanedione. The loss of haemolysis showed strong correlation with the number of Lys or Arg residues modified. CD analyses, however, showed that the conformation of SNTX was not significantly affected by these chemical modifications. Further, the haemolytic activity of SNTX was competitively inhibited by various negatively charged lipids, such as phosphatidylserine, cardiolipin and monosialogangliosides. These results indicate that SNTX induces potent haemolytic activity through the formation of pores in the cell membrane; and that cationic residues play a crucial role in its cytolytic mechanism.