The Dual Activity Responsible for the Elongation and Branching of β-(1,3)-Glucan in the Fungal Cell Wall.
The Dual Activity Responsible for the Elongation and Branching of β-(1,3)-Glucan in the Fungal Cell Wall.
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DOI：
10.1128/mbio.00619-17
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发表时间：
2017-06-20
期刊：
影响因子：
6.4
通讯作者：
Latge JP
中科院分区：
文献类型：
--
作者：
Aimanianda V;Simenel C;Garnaud C;Clavaud C;Tada R;Barbin L;Mouyna I;Heddergott C;Popolo L;Ohya Y;Delepierre M;Latge JP
β-（1，3）-葡聚糖是真菌细胞壁的主要成分，通过β-（1，6）-糖苷键衍生，促进其与其他细胞壁成分的结合，有助于细胞壁的正确组装。以酿酒酵母为模型，我们开发了一种定量β-（1，3）-葡聚糖上β-（1，6）-分支的方案。透化的酿酒酵母和放射性标记的底物UDP-（14 C）葡萄糖使我们能够确定分支动力学。旨在鉴定其中具有减少的分支的缺失突变体的筛选仅揭示了两个，bgl 2 Δ和gas 1 Δ突变体，与野生型菌株相比，分别显示分支减少15%和70%。有趣的是，重组Gas 1 p在具有β-（1，3）-延伸酶活性后，在β-（1，3）-寡聚体上引入了β-（1，6）-分支。Gas 1 p的连续延伸和分支活性发生在线性β-（1，3）-寡聚物以及Bgl 2 p催化的产物[通过线性β-（1，6）-键连接的短β-（1，3）-寡聚物]上。双酿酒酵母gas 1 Δ bgl 2 Δ突变体表现出严重的病态表型。来自烟曲霉的ScGas 1 p直向同源物Gel 4p也显示出双重β-（1，3）-葡聚糖延伸和分支活性。ScGas 1 p和烟曲霉Gel 4p序列都具有碳水化合物结合模块（CBM），CBM 43，其是双重β-（1，3）-葡聚糖延伸和分支活性所需的。我们的报告揭示了β-（1，3）-葡聚糖分支机制，这是一种在真菌生命所必需的细胞壁构建过程中发生的现象。真菌细胞壁对于生长、形态发生、保护和存活是必不可少的。尽管细胞壁生物发生是必不可少的，但人们对核心β-（1，3）-葡聚糖衍生物的了解很少;衍生的β-（1，3）-葡聚糖与其他细胞壁组分相互连接。一旦线性β-（1，3）-葡聚糖被质膜结合的葡聚糖合成酶合成，随后的事件是其在细胞壁空间中的分支事件。以酿酒酵母为模型，我们分别鉴定了GH 72和GH 17家族糖基转移酶Gas 1 p和Bgl 2 p，它们参与β-（1，3）-葡聚糖分支。双Scgas 1 Δ bgl 2 Δ突变体的病态表型表明β-（1，3）-葡聚糖分支是必需的。除了ScGas 1 p之外，GH 72家族ScGas 2 p和烟曲霉Gel 4p（在它们的序列中具有CBM 43）显示双重β-（1，3）-葡聚糖延伸和分支活性。我们的报告确定了真菌细胞壁β-（1，3）-葡聚糖分支机制。β-（1，3）-葡聚糖分支的重要性表明，参与葡聚糖分支的酶可用作抗真菌靶标。
β-(1,3)-Glucan, the major fungal cell wall component, ramifies through β-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. Using Saccharomyces cerevisiae as a model, we developed a protocol to quantify β-(1,6)-branching on β-(1,3)-glucan. Permeabilized S. cerevisiae and radiolabeled substrate UDP-(14C)glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the bgl2Δ and gas1Δ mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced β-(1,6)-branching on the β-(1,3)-oligomers following its β-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear β-(1,3)-oligomers as well as Bgl2p-catalyzed products [short β-(1,3)-oligomers linked by a linear β-(1,6)-linkage]. The double S. cerevisiae gas1Δ bgl2Δ mutant showed a drastically sick phenotype. An ScGas1p ortholog, Gel4p from Aspergillus fumigatus, also showed dual β-(1,3)-glucan elongating and branching activity. Both ScGas1p and A. fumigatus Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual β-(1,3)-glucan elongating and branching activity. Our report unravels the β-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life. The fungal cell wall is essential for growth, morphogenesis, protection, and survival. In spite of being essential, cell wall biogenesis, especially the core β-(1,3)-glucan ramification, is poorly understood; the ramified β-(1,3)-glucan interconnects other cell wall components. Once linear β-(1,3)-glucan is synthesized by plasma membrane-bound glucan synthase, the subsequent event is its branching event in the cell wall space. Using Saccharomyces cerevisiae as a model, we identified GH72 and GH17 family glycosyltransferases, Gas1p and Bgl2p, respectively, involved in the β-(1,3)-glucan branching. The sick phenotype of the double Scgas1Δ bgl2Δ mutant suggested that β-(1,3)-glucan branching is essential. In addition to ScGas1p, GH72 family ScGas2p and Aspergillus fumigatus Gel4p, having CBM43 in their sequences, showed dual β-(1,3)-glucan elongating and branching activity. Our report identifies the fungal cell wall β-(1,3)-glucan branching mechanism. The essentiality of β-(1,3)-glucan branching suggests that enzymes involved in the glucan branching could be exploited as antifungal targets.