Accelerated Evolution of Venom Gland Isozymes

蛇毒含有多种生理活性酶，对哺乳动物靶分子具有高选择性作用。蛇知道哺乳动物（人类）体内平衡的一切。最有趣的问题之一是蛇毒素是如何进化到获得哺乳动物特有的功能的。近年来，我们发现蛇磷脂酶A_2 （PLA_2）同工酶基因仅在成熟的蛋白质编码区加速进化而获得多种生理功能。本项目的一个主要主题是在其他蛇类毒素和物种中证明这一假设的普遍性，以阐明适应性进化机制。首先，我们从Naja Naja kaouthia和Dendroaspis angnsticeps（绿曼巴）毒液腺中制备cDNA文库。从中华水貂蛇毒腺中获得了50个编码I组PLA_2同工酶cdna的阳性克隆。对两个cDNA克隆进行测序。它们的核苷酸序列编码新的PLA_2同工酶，彼此之间有4个氨基酸差异。从蛇毒腺cDNA文库中克隆出4个编码“三指”型同工毒素的新cDNA，并对它们进行了测序。对“三指”型异毒素cdn核苷酸序列的比较表明，成熟蛋白编码区比非编码区具有更大的变异性，且成熟蛋白编码区每非同义位点的核苷酸取代数（K_A）接近或大于每同义位点的核苷酸取代数（K_S）。这些观察结果表明，这些同工毒素也是通过达尔文式加速进化进化而来的。显然，加速进化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.