Molecular cloning, phylogenetic analysis and three-dimensional modeling of Cu,Zn superoxide dismutase (CnSOD1) from three varieties of Cryptococcus neoformans.
Molecular cloning, phylogenetic analysis and three-dimensional modeling of Cu,Zn superoxide dismutase (CnSOD1) from three varieties of Cryptococcus neoformans.
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DOI：
10.1016/s0378-1119(01)00408-5
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发表时间：
2001-05
期刊：
影响因子：
3.5
通讯作者：
S. Chaturvedi;Andrew Hamilton;Paul Hobby;Guan Zhu;Charles V. Lowry;Vishnu Chaturvedi;Vishnu Chaturvedi;Vishnu Chaturvedi
中科院分区：
文献类型：
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作者：
S. Chaturvedi;Andrew Hamilton;Paul Hobby;Guan Zhu;Charles V. Lowry;Vishnu Chaturvedi;Vishnu Chaturvedi;Vishnu Chaturvedi
新型隐球菌（Cryptococcusneoformans，Cn）是引起真菌性脑膜脑炎的病原菌，有3个变种，不同的宿主有不同的偏好。为了探讨这些致病差异的机制，我们已经确定了Cu，Zn SOD基因（CnSOD 1）。用Cn var.格鲁比酵母表达文库互补克隆的ORF为462 bp，推导的154 aa序列与S.soaeSOD1的同源性为61%，与其它真核SOD 1的同源性为53-65%。Cn var.用grubiiiCnSOD 1 cDNA克隆了grubiiCnSOD 1的cDNA。neoformans和var.加提。三个品种的ORF在推导的氨基酸上显示了20-29%的差异，其中Cn var. Grubii和Cn var.加提。通过粘粒文库筛选和PCR克隆获得SOD 1基因组，该基因组由5个位置相同的内含子和一个典型的5′剪接点序列GTNNGY组成。这些内含子也表现出较大的nt变异的三个Cn品种。系统发育分析显示，CnSOD 1是在一组不同的其他真核SOD 1和一个显着的分歧的变种。格鲁比湾加提。CnSOD 1推导的蛋白质建模的基础上S.党aeSOD 1的晶体结构，表现出良好的拟合。大多数非同义氨基酸替换发生在分子的外部，这些可能有助于三个品种之间的抗原性差异。值得注意的是，Cn var. neoformans和var. Gattii Cu，Zn SOD中有三个甘氨酸取代位点（Gly 26，Gly 92和Gly 123分别取代Asn 26，Ser 92和Ser 123），这可能是该酶相对维斯Cn var. grubii。这是第一次从三个Cn品种的蛋白质编码基因的核苷酸和结构比较，这可能为未来的研究提供了一个框架，Cu，Zn SOD在Cn发病机制中的作用。
Cryptococcusneoformans (Cn), causal agent of fungal meningoencephalitis, has three varieties with variable host predilection. To explore mechanisms for these pathogenic differences, we have characterized Cu,Zn SOD gene (CnSOD1). A Saccharomycescerevisiaesod1Δ mutant was complemented with Cn var. grubii yeast expression library. The complementing clone had an ORF of 462 bp and the deduced 154 aa sequence showed 61% identity with S.cerevisiaeSOD1 and 53–65% with other eukaryotic SOD1s. Cn var. grubiiCnSOD1 cDNA was used to clone corresponding cDNAs from var. neoformans and var. gattii. ORFs from three varieties revealed 20–29% differences in deduced aa (s) with a significant 6% non-synonymous aa substitution between Cn var. grubii and Cn var. gattii. Cosmid library screening and PCR cloning were used to obtain genomic SOD1, which was split by five introns with identical placements and a typical 5′ splice junction sequence, GTNNGY. These introns also showed a large nt variation among the three Cn varieties. Phylogenetic analyses revealed CnSOD1 to be in a group distinct from other eukaryotic SOD1s and with a significant divergence of the var. grubii from var. gattii. The CnSOD1 -deduced protein was modeled based on the crystal structure of S.cerevisiaeSOD1, which showed an excellent fit. Most of the non-synonymous aa substitutions occurred on the outside of the molecule and these may contribute to differences in antigenicity among the three varieties. Notably, Cn var. neoformans and var. gattii Cu,Zn SOD had three substitutions of glycine (Gly26, Gly92 and Gly123 for Asn26, Ser92 and Ser123) that may contribute to the observed lower thermostability of this enzyme vis-a-vis Cn var. grubii. This is the first nucleotide and structural comparison of a protein-encoding gene from the three Cn varieties, which may provide a framework for future studies on the role of Cu,Zn SOD in Cn pathogenesis.