Accelerated Evolution of Venom Gland Isozymes

蛇毒中含有多种生理活性酶，它们高选择性地作用于哺乳动物的靶分子。蛇对哺乳动物(人类)动态平衡了如指掌。最耐人寻味的问题之一是，蛇毒素是如何进化出来的，以获得哺乳动物特有的功能。近年来，我们发现蛇磷脂酶A_2(PLA2)同工酶基因只在成熟的蛋白编码区通过加速进化获得了不同的生理功能。本课题的一个主要主题是证明这一假说对其他蛇毒素和物种的普适性，以阐明其适应性进化机制。从中华眼镜蛇毒腺体中获得50个编码第I组磷脂酶A_2同工酶的阳性克隆。对两个克隆进行了测序。它们的核苷酸序列编码新的PLA2同工酶，它们之间有4个氨基酸的差异。从弓形虫毒腺的cDNA文库中，克隆了4个新的编码“那里指状”同工毒素的cDNA，它们也具有不同的功能。“三指型”同源毒素cDAN的核苷酸序列比较表明，成熟蛋白编码区的变异比非编码区大得多，并且成熟蛋白编码区每个非同义位点的核苷酸替换数(K_A)接近或大于每个同义位点的核苷酸替换数(K_S)。这些观察表明，这些同种毒素也是通过达尔文式的加速进化而进化的。显然，加速降解在蛇毒同工酶或同工蛋白家族中是普遍存在的。

Snake venom contains a variety of physiological active enzymes which act on mammalian target molecules with high selectivety. Snakes know everything about mammalian (human) homeostasis. One of the most intriguing questions is how snake toxins have evolved to acquire such functions specific to mammalian constitutes. Recently, we discovered that crotalidae snake phospholipase A_2 (PLA_2) isozyme genes have evolved via accelerated evolution only in the mature protein-coding region to gain diverse physiological functions.A major theme in this project is to prove the universality of this hypothesis for other snake toxins and species to elucidate the adaptive evolutionary mechanism.First, we prepared cDNA libraries from Naja naja kaouthia and Dendroaspis angnsticeps (green mamba) venom gland. We obtained 50 positive clones encoding group I PLA_2 isozyme cDNAs from Naja naja kaouthia venom gland. Two cDNA clones were seqenced. Their nucleotide sequences encoded new PLA_2 isozymes with four amino acid differences to each other.From D.angnsticeps venom gland cDNA library, four new cDNAs encoding "theree finger" shaped isotoxins, which also exhibit various diverse fanctions, were cloned and sequenced. The comparison of the nucleotide sequences of the "three finger" shaped isotoxin cDANs revealed that the mature protein-coding region is much more variable than the noncoding region and that the numbers of nucleotide substitutions per nosnsynonymous site (K_A) are close to or larger than the numbers of nucleotide substitutions per synonymous site (K_S) in the mature protein-coding region. These observations suggest that these isotoxins have also evolved via Darwinian-type accelerated evolution. It is apparent that accelerated eolution is universal in snake venom isozyme or isoprotein families.