OCTOPUS S-CRYSTALLINS WITH ENDOGENOUS GLUTATHIONE-S-TRANSFERASE (GST) ACTIVITY - SEQUENCE COMPARISON AND EVOLUTIONARY RELATIONSHIPS WITH AUTHENTIC GST ENZYMES
OCTOPUS S-CRYSTALLINS WITH ENDOGENOUS GLUTATHIONE-S-TRANSFERASE (GST) ACTIVITY - SEQUENCE COMPARISON AND EVOLUTIONARY RELATIONSHIPS WITH AUTHENTIC GST ENZYMES
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DOI：
10.1042/bj3090793
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发表时间：
1995-08-01
影响因子：
4.1
通讯作者：
CHANG, GG
中科院分区：
文献类型：
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作者：
CHIOU, SH;YU, CW;CHANG, GG
S晶体蛋白是头足类动物(章鱼和鱿鱼)晶状体中的一种主要蛋白质。为了便于克隆该晶体蛋白基因，从章鱼晶状体的Poly(A)(+)mRNA中构建了cDNA，并通过聚合酶链式反应(PCR)扩增，进行核苷酸序列测定。对编码该晶体蛋白的15个阳性克隆中的10个进行了测序，发现有3个不同的S-晶体蛋白亚型，核苷酸序列同源性为61%-，氨基酸序列同源性为42%-58%。这些电荷异构体晶体蛋白与谷胱甘肽S转移酶的氨基酸序列同源性在26%到33%之间，谷胱甘肽转移酶是大多数哺乳动物组织中存在的一种主要解毒酶。为了进一步分析，在细菌表达系统PQE-30中表达了一个S晶体蛋白基因，并对在大肠杆菌中表达的S晶体蛋白进行了纯化，以确定其酶学性质。我们发现，表达的章鱼S晶体蛋白的GST活性明显低于其他组织的GST。对鱿鱼和章鱼晶状体中的S晶体蛋白和不同类别的GST进行序列比较和系统发育树的构建表明，S晶体蛋白是一个多基因家族，在结构上与Alpha类GST有亲缘关系，可能是由祖先的GST通过基因复制和随后的多次突变替换而来的。
S-Crystallin is a major protein present in the lenses of cephalopods (octopus and squid). To facilitate the cloning of this crystallin gene, cDNA was constructed from the poly(A)(+) mRNA of octopus lenses, and amplified by PCR for nucleotide sequencing. Sequencing of 10 of 15 positive clones coding for this crystallin revealed three distinct S-crystallin isoforms with 61-64% identity in nucleotide sequences and 42-58% similarity in amino acid sequences when compared with homologous crystallins in squid lenses. These charge-isomeric crystallins also show between 26 and 33% amino acid sequence identity to four major classes of glutathione S-transferase (GST), a major detoxification enzyme present in most mammalian tissues. For further analysis, expression of one of the S-crystallin cDNAs was carried out in the bacterial expression system pQE-30, and the S-crystallin protein produced in Escherichia coli was purified to homogeneity to determine the enzymic properties. We found that the expressed octopus S-crystallin possessed much lower GST activity than the authentic GSTs from other tissues. Sequence comparison and construction of phylogenetic trees for S-crystallins from squid and octopus lenses and various classes of GSTs revealed that S-crystallins represent a multigene family which is structurally related to Alpha-class GSTs and probably derived from the ancestral GST by gene duplication and subsequent multiple mutational substitutions.