Chioato Lucimara, De Oliveira Arthur H C, Ruller Roberto, Sá Juliana M, Ward Richard J
Departamento de Bioqui;mica e Imunologia, FMRP-USP, Universidade de São Paulo, Brazil.
Biochem J. 2002 Sep 15;366(Pt 3):971-6. doi: 10.1042/BJ20020092.
Bothropstoxin-I (BthTx-I) is a Lys(49)-phospholipase A(2) from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca(2+)-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117-122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys(122), these same substitutions did not significantly alter the Ca(2+)-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca(2+)-independent membrane-damaging activity but, with the exception of Lys(122), had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115-119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A(2), and support a model of a Ca(2+)-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.
矛头蝮毒素-I（BthTx-I）是一种来自巴西矛头蝮蛇毒液的Lys(49)-磷脂酶A(2)，具有肌毒性和不依赖Ca(2+)的膜损伤活性。这些活性的结构决定因素尚不明确，因此已使用定点诱变来替换该蛋白质C端环区域中第115至129位之间的所有阳离子和芳香族残基。在117-122区域用丙氨酸取代赖氨酸和精氨酸残基导致重组BthTx-I的肌毒性活性显著降低。除了Lys(122)外，这些相同的取代并没有显著改变不依赖Ca(2+)的膜损伤活性。相反，在第115、116和122位取代带正电荷的残基导致不依赖Ca(2+)的膜损伤活性降低，但除了Lys(122)外，对肌毒性没有影响。这些结果表明这两种活性是独立的，并且由C端环中离散但部分重叠的基序决定。对该蛋白质同一部分芳香族残基进行定点诱变的结果表明，包括第115-119位残基的区域与膜界面有表面相互作用，并且第125位附近的残基部分插入脂质膜中。这些结果代表了磷脂酶A(2)中肌毒性位点的首次详细定位，并支持了一种不依赖Ca(2+)的膜损伤机制模型，其中BthTx-I的C端区域与磷脂双层相互作用并导致其扰动。
