Grobler J A, Rao K R, Pervaiz S, Brew K
Department of Biochemistry and Molecular Biology, University of Miami, Florida 33136.
Arch Biochem Biophys. 1994 Sep;313(2):360-6. doi: 10.1006/abbi.1994.1399.
The amino acid sequences of two canine lysozymes, from milk and spleen, have been elucidated by direct sequence analyses of the purified proteins and fragments generated from them. The two enzymes are highly divergent, differing from each other by 45% in sequence, but each is closely similar to lysozymes previously obtained from other mammalian species. The milk lysozyme is similar in sequence to equine and donkey milk lysozymes (83% identity) and, like these enzymes, contains a bound Ca2+ ion while the spleen enzyme is most similar in sequence to the majority of previously studied mammalian and avian lysozymes (80 to 83% identity) and, based on its sequence, does not contain a Ca(2+)-binding site. This demonstrates that Ca(2+)-binding lysozymes are expressed in at least two mammalian orders, the carnivores and perissodactyls, as well as confirming that the genes for the Ca(2+)-binding and conventional lysozymes are paralogous. The latter point was further confirmed by the isolation and partial sequence analysis of a conventional lysozyme from equine spleen. The relationships of these new lysozyme sequences to those of other lysozymes and their homologues, the alpha-lactalbumins, were analyzed using different molecular phylogeny algorithms, producing a new model for the evolutionary origins of the superfamily. The most significant conclusion to be drawn from this model is that Ca(2+)-binding activity was an ancient feature of this protein superfamily which was lost during the evolutionary development of the conventional lysozymes. It also supports a previous suggestion that the alpha-lactalbumins and lysozymes diverged at a time earlier than the divergence of the fishes and tetrapods.
通过对纯化蛋白及其产生的片段进行直接序列分析，已阐明了两种犬溶菌酶（来自牛奶和脾脏）的氨基酸序列。这两种酶高度不同，序列差异达45%，但每种都与先前从其他哺乳动物物种获得的溶菌酶非常相似。牛奶溶菌酶在序列上与马和驴的牛奶溶菌酶相似（同一性为83%），并且像这些酶一样，含有一个结合的Ca2+离子，而脾脏酶在序列上与大多数先前研究的哺乳动物和鸟类溶菌酶最相似（同一性为80%至83%），基于其序列，不含有Ca(2+)结合位点。这表明Ca(2+)结合溶菌酶至少在两个哺乳动物目（食肉目和奇蹄目）中表达，同时也证实了Ca(2+)结合溶菌酶和传统溶菌酶的基因是旁系同源的。从马脾脏中分离出一种传统溶菌酶并进行部分序列分析，进一步证实了后一点。使用不同的分子系统发育算法分析了这些新的溶菌酶序列与其他溶菌酶及其同源物α-乳白蛋白序列的关系，产生了一个关于该超家族进化起源的新模型。从该模型得出的最重要结论是，Ca(2+)结合活性是该蛋白质超家族的一个古老特征，在传统溶菌酶的进化发展过程中丧失了。它还支持了先前的一个观点，即α-乳白蛋白和溶菌酶在鱼类和四足动物分化之前就已经分化。
