Inhibition Mechanism of a Novel Phospholipase A_2 Inhibitor from the Blood of the Venomous Snakes

1.中国乳鼠血浆中新型磷脂酶A_2抑制剂的纯化我们从中国乳鼠血浆中分离纯化了三种不同类型的磷脂酶A_2抑制剂(PLIpha、PLIbeta和PLIGamma)。PLIpha被发现是我们已经报道的CRD样抑制剂，选择性地抑制II组酸性PLA2。PLIbeta和PLIGamma是本研究中提纯的新型抑制剂。前者选择性地抑制第II组基础磷脂酶A_2s，后者同样抑制包括第I组磷脂酶A_2s、第II组磷脂酶A_2s和蜜蜂磷脂酶A_2(第III组)在内的所有磷脂酶A_2。中国乳鼠肝脏磷脂酶A_2抑制物基因的克隆我们构建了中国乳鼠肝脏基因的cDNA文库，克隆了PLIpha、PLIbeta和PLIGamma的基因，并测定了它们的核苷酸序列。结果发现，PLIGamma与已从泰国眼镜蛇血浆和海蛇血浆中纯化的抑制剂类型相同，具有与Ly-6超家族蛋白相似的半胱氨酸残基模式。另一方面，PLIbeta与人血清中富含亮氨酸的α_2糖蛋白具有较高的序列同源性。PLIbeta的一个分子由三个相同的亚基组成，它与三个PLA2分子结合。通过对PLIbeta特异性的比较，推测位于第二组PLA2界面结合面或其附近的氨基酸残基参与了与PLIbeta的选择性结合。另一方面，位于磷脂酶A2的Ca^&lt；2+&gt；结合环或其附近的残基可能参与了与PLIGamma的结合。四毒蛇血清中磷脂酶A_2抑制物的纯化在无毒蛇的血清中发现了磷脂酶A_2抑制剂，并对其进行了纯化。纯化后的抑制物为相同类型的PLIGamma。

1. Purification of novel phospholipase A_2 inhibitors from the blood plasma of Chinese mamushiWe have purified three distinct types of phospholipase A_2 inhibitors (PLIalpha, PLIbeta, and PLIgamma) from the blood plasma of Chinese mamushi. PLIalpha was found to be CRD-like inhibitor that we had already reported to inhibit selectively the group-II acidic PLA_2s. PLIbeta and PLIgamma were novel inhibitors purified in the present study. The former inhibited selectively the group-II basic PLA_2s, while the latter inhibited equally all of the PLA_2s including group-I PLA_2s, group-II PLA_2s, and honey bee PLA_2 (group-III).2. cDNA cloning of Chinese mamushi PLA_2 inhibitorsWe have constructed cDNA library for the liver mRNA of Chinese mamushi, cloned cDNA for PLIalpha, PLIbeta, and PLIgamma, and determined their nucleotide sequences. As a results, PLIgamma was found to be the same type of the inhibitors that had been already purified from Thailand cobra plasma and sea snake plasma, having the similar pattern of cysteine residues to those of the proteins belonging to Ly-6 superfamily. On the other hand, PLIbeta was found to have high sequence homology to leucine-rich alpha_2 glycoprotein from human sera.3. Interaction between PLA_2 and inhibitorOne molecule of PLIbeta was composed of three identical subunits and it bound three PLA_2 molecules. From the comparisons of the specificity of PLIbeta, some amino acid residues located in or close to the interfacial binding surface of the group-II PLA_2 were suggested to be involved in the selective binding to PLIbeta. On the other hand, the residues located in or close to the Ca^<2+> binding loop of PLA_2 were suggested to be involved in the binding to PLIgamma.4. Purification of PLA_2 inhibitor from the sera of Elaphe quadrivirgataPLA_2 inhibitors were found to be present in the serum of nonvenomous snakes, Elaphe quadrivirgata, and were purified. The purified inhibitor was found to be the same type of PLIgamma.