Identification of Two Mannosyltransferases Contributing to Biosynthesis of the Fungal-type Galactomannan α-Core-Mannan Structure in Aspergillus fumigatus.
Identification of Two Mannosyltransferases Contributing to Biosynthesis of the Fungal-type Galactomannan α-Core-Mannan Structure in Aspergillus fumigatus.
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DOI：
10.1038/s41598-018-35059-2
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发表时间：
2018-11-16
影响因子：
4.6
通讯作者：
Oka T
中科院分区：
文献类型：
--
作者：
Onoue T;Tanaka Y;Hagiwara D;Ekino K;Watanabe A;Ohta K;Kamei K;Shibata N;Goto M;Oka T
真菌型半乳甘露聚糖（FTGM）是一种由α-(1→2)-/α-(1→6)-甘露糖基残基和β-(1→5)-/β-(1→6)-半乳甘露糖基残基组成的多糖，存在于人致病性真菌烟曲霉的外细胞壁中。FTGM含有线性α-甘露聚糖结构，称为核心甘露聚糖，由9或10个α-(1→2)-甘露聚糖单元由α-（1→6）键连接而成。然而，参与甘露聚糖核心生物合成的酶仍然未知。我们推测烟曲霉中两个α-1,2-甘露糖基转移酶基因Afu5g02740/AFUB_051270（这里称为核心甘露糖合成酶A [CmsA]）和Afu5g12160/AFUB_059750 （CmsB）参与了FTGM核心甘露糖的生物合成。我们构建了CmsA重组蛋白，并利用化学合成的底物对硝基苯α-d-甘露糖吡喃苷作为受体，检测了甘露糖基转移酶的活性。对CmsA酶解产物的分析表明，CmsA具有将甘露糖苷转移到α-甘露糖的C-2位置的能力。CmsA还能将甘露糖残渣转化为α-(1→2)-甘露糖糖和α-(1→6)-甘露糖糖，对α-(1→6)-甘露糖糖的比活性比α-(1→2)-甘露糖糖高31倍。分离FTGM的质子核磁共振（1H-NMR）波谱和凝胶过滤层析显示，在烟曲霉菌株∆cmsA、∆cmsB和∆cmsA∆cmsB中，核心甘露聚糖结构发生了显著改变和缩短。cmsA或cmsB的破坏导致菌丝伸展受到严重抑制，菌丝分支异常，菌丝形成球囊结构，分生孢子形成减少。通过cmsA和cmsB的补充，恢复了正常野生型甘露糖核的结构和发育表型。这些发现表明，α-1,2-甘露糖基转移酶CmsA和假定的甘露糖基转移酶CmsB都参与了FTGM的生物合成。
Fungal-type galactomannan (FTGM) is a polysaccharide composed of α-(1 → 2)-/α-(1 → 6)-mannosyl and β-(1 → 5)-/β-(1 → 6)-galactofuranosyl residues located at the outer cell wall of the human pathogenic fungus Aspergillus fumigatus. FTGM contains a linear α-mannan structure called core-mannan composed of 9 or 10 α-(1 → 2)-mannotetraose units jointed by α-(1 → 6)-linkages. However, the enzymes involved in core-mannan biosynthesis remain unknown. We speculated that two putative α-1,2-mannosyltransferase genes in A. fumigatus, Afu5g02740/AFUB_051270 (here termed core-mannan synthase A [CmsA]) and Afu5g12160/AFUB_059750 (CmsB) are involved in FTGM core-mannan biosynthesis. We constructed recombinant proteins for CmsA and detected robust mannosyltransferase activity using the chemically synthesized substrate p-nitrophenyl α-d-mannopyranoside as an acceptor. Analyses of CmsA enzymatic product revealed that CmsA possesses the capacity to transfer a mannopyranoside to the C-2 position of α-mannose. CmsA could also transfer a mannose residue to α-(1 → 2)-mannobiose and α-(1 → 6)-mannobiose and showed a 31-fold higher specific activity toward α-(1 → 6)-mannobiose than toward α-(1 → 2)-mannobiose. Proton nuclear magnetic resonance (1H-NMR) spectroscopy and gel filtration chromatography of isolated FTGM revealed that core-mannan structures were drastically altered and shortened in disruptant A. fumigatus strains ∆cmsA, ∆cmsB, and ∆cmsA∆cmsB. Disruption of cmsA or cmsB resulted in severely repressed hyphal extension, abnormal branching hyphae, formation of a balloon structure in hyphae, and decreased conidia formation. The normal wild type core-mannan structure and developmental phenotype were restored by the complementation of cmsA and cmsB in the corresponding disruptant strains. These findings indicate that both CmsA, an α-1,2-mannosyltransferase, and CmsB, a putative mannosyltransferase, are involved in FTGM biosynthesis.
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影响因子：
3.6
作者：
Komachi Y;Hatakeyama S;Motomatsu H;Futagami T;Kizjakina K;Sobrado P;Ekino K;Takegawa K;Goto M;Nomura Y;Oka T
通讯作者：
Oka T
影响因子：
3.4
作者：
Henry, Christine;Fontaine, Thierry;Latge, Jean-Paul
通讯作者：
Latge, Jean-Paul
影响因子：
2.8
作者：
Jiang, Hechun;Ouyang, Haomiao;Jin, Cheng
通讯作者：
Jin, Cheng
影响因子：
4.8
作者：
Engel, Jakob;Schmalhorst, Philipp S.;Routier, Francoise H.
通讯作者：
Routier, Francoise H.
影响因子：
4.3
作者：
Kudoh, Atsushi;Okawa, Yoshio;Shibata, Nobuyuki
通讯作者：
Shibata, Nobuyuki