Dolichol phosphate mannose synthase from the pathogenic yeast Candida albicans is a multimeric enzyme
Dolichol phosphate mannose synthase from the pathogenic yeast Candida albicans is a multimeric enzyme
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DOI：
10.1016/j.bbagen.2015.08.012
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发表时间：
2015-11-01
影响因子：
3
通讯作者：
Palamarczyk, Grazyna
中科院分区：
文献类型：
--
作者：
Juchimiuk, Mateusz;Kruszewska, Joanna;Palamarczyk, Grazyna
背景：多萜醇磷酸甘露糖合成酶（DPMS）是N - 和O - 连接糖基化以及糖基磷脂酰肌醇（GPI）锚定合成中的关键酶。DPMS通过将甘露糖基残基从GDP - 甘露糖转移至多萜醇磷酸来生成DPM，DPM是上述过程的底物。在此我们描述DPMS在白色念珠菌生理学中的作用，重点是细胞壁组成和形态发生。
方法：克隆白色念珠菌DPMS亚基的基因，进行标记并在酿酒酵母中表达。构建并分析具有可控DPM基因表达的白色念珠菌菌株。使用逆转录聚合酶链反应（RT - PCR）和放射性底物测量基因表达和酶活性。用微量稀释法测试对化学试剂的敏感性。通过高效液相色谱（HPLC）评估细胞壁的组成。通过蛋白质印迹法分析标记蛋白的糖基化状态。在促进菌丝和厚垣孢子形成的培养基上检查菌株的形态分化。
结果：我们证明白色念珠菌DPMS由三个相互作用的亚基组成，其中Dpm1和Dpm3是必不可少的，而Dpm2可提高酶活性。DPMS基因表达降低导致DPMS活性下降，对细胞壁干扰剂的敏感性增加以及细胞壁组成改变。突变体Tetp - DPM1和Tetp - DPM3显示蛋白质糖基化缺陷，并且在菌丝和厚垣孢子形成方面受损。
主要结论：与酿酒酵母相反，白色念珠菌的DPMS属于具有多聚体蛋白质结构的DPMS家族。
一般意义：这项工作提供了关于致病性酵母白色念珠菌正确蛋白质糖基化和形态发生所需因素的重要数据。（C）2015爱思唯尔公司。保留所有权利。
Background: Dolichol phosphate mannose synthase (DPMS) is a key enzyme in N- and O-linked glycosylations and glycosylphosphatidylinositol (GPI)-anchor synthesis. DPMS generates DPM, the substrate for mentioned processes, by the transfer of mannosyl residue from GDP-Man to dolichol phosphate. Here we describe the role of DPMS for Candida albicans physiology with emphasis on the cell wall composition and morphogenesis.Methods: C. albicans genes for DPMS subunits were cloned, tagged and expressed in Saccharomyces cerevisiae. The C. albicans strains with controlled expression of DPM genes were constructed and analyzed. Gene expression and enzyme activities were measured using RT-PCR and radioactive substrate. Sensitivities against chemical agents were tested with microdilution method. The composition of the cell wall was estimated by HPLC. Glycosylation status of the marker protein was analyzed by Western blot. Morphological differentiation of the strains was checked on the media promoting hyphae and chlamydospore formation.Results: We demonstrate that C. albicans DPMS consists of three interacting subunits, among which Dpm1 and Dpm3 are indispensable, whereas Dpm2 increases enzymatic activity. Lowered expression of DPMS genes results in decreased DPMS activity, increased susceptibility to cell wall perturbing agents and in altered cell wall composition. Mutants Tetp-DPM1 and Tetp-DPM3 show defective protein glycosylation and are impaired in hyphae and chlamydospore formation.Major conclusion: DPMS from C. albicans, opposite to S. cerevisiae, belongs to the family of DPMS with multimeric protein structure.General significance: This work provides important data about factors required for a proper protein glycosylation and for morphogenesis of pathogenic yeast C. albicans. (C) 2015 Elsevier B.V. All rights reserved.