Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto, Japan.
Department of Infection and Host Defense, Tohoku Medical and Pharmaceutical University, Sendai, Japan.
Sci Rep. 2018 Nov 16;8(1):16918. doi: 10.1038/s41598-018-35059-2.
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 (H-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.
真菌型半乳甘露聚糖（FTGM）是一种多糖，由位于人类致病真菌烟曲霉的细胞壁外层的α-(1→2)-/α-(1→6)-甘露糖基和β-(1→5)-/β-(1→6)-半乳糖呋喃糖基残基组成。FTGM 含有一种线性的α-甘露聚糖结构，称为核心甘露聚糖，由 9 或 10 个通过α-(1→6)-键连接的α-(1→2)-甘露四糖单元组成。然而，核心甘露聚糖生物合成中涉及的酶仍然未知。我们推测烟曲霉中的两个假定的α-1,2-甘露糖基转移酶基因，Afu5g02740/AFUB_051270（称为核心甘露聚糖合酶 A [CmsA]）和 Afu5g12160/AFUB_059750（CmsB）参与 FTGM 核心甘露聚糖的生物合成。我们构建了 CmsA 的重组蛋白，并使用化学合成的供体 p-硝基苯-α-D-甘露吡喃糖苷检测到了强大的甘露糖基转移酶活性。对 CmsA 酶促产物的分析表明，CmsA 能够将甘露吡喃糖苷转移到α-甘露糖的 C-2 位。CmsA 还可以将甘露糖残基转移到α-(1→2)-甘露二糖和α-(1→6)-甘露二糖上，并且对α-(1→6)-甘露二糖的比活性比α-(1→2)-甘露二糖高 31 倍。分离的 FTGM 的质子核磁共振（H-NMR）光谱和凝胶过滤色谱表明，核心甘露聚糖结构在缺失突变株 A. fumigatus 菌株 ∆cmsA、∆cmsB 和 ∆cmsA∆cmsB 中被严重改变和缩短。cmsA 或 cmsB 的缺失导致菌丝延伸严重受到抑制、异常分支菌丝、菌丝形成气球结构以及分生孢子形成减少。在相应的缺失突变株中，cmsA 和 cmsB 的互补恢复了正常野生型核心甘露聚糖结构和发育表型。这些发现表明，CmsA（一种α-1,2-甘露糖基转移酶）和 CmsB（一种假定的甘露糖基转移酶）都参与了 FTGM 的生物合成。
