Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan.
Carbohydr Res. 2010 Nov 22;345(17):2516-22. doi: 10.1016/j.carres.2010.09.006. Epub 2010 Sep 9.
Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have β-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as l-Ara and GlcA are attached. For the present study, an α-L-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-β-(1→6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing α-L-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on β-(1→6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, β-(1→6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa.
阿拉伯半乳聚糖蛋白(AGPs)是一类植物糖蛋白,其核心蛋白上连接有较大的碳水化合物侧链。AGP 的碳水化合物侧链通常以β-(1→3)(1→6)-半乳糖作为主链,其上连接有其他辅助糖,如 l-Ara 和 GlcA。在本研究中,从粗糙脉孢菌(Neurospora crassa)中鉴定出属于糖苷水解酶家族(GHF)54 的α-L-阿拉伯呋喃糖苷酶 NcAraf1 和 GHF 5 的内切-β-(1→6)-半乳糖酶 Nc6GAL。在巴斯德毕赤酵母中表达的重组 NcAraf1(rNcAraf1)可水解萝卜 AGPs 以及阿拉伯聚糖和阿拉伯木聚糖,对含有α-L-阿拉伯呋喃糖基残基的多糖表现出相对较广的底物特异性。在巴斯德毕赤酵母中表达的重组 Nc6GAL(rNc6GAL)特异性作用于β-(1→6)-半乳糖基残基。虽然萝卜根 AGP 单独用 rNc6GAL 几乎不能水解,但 rNcAraf1 和 rNc6GAL 的组合可将β-(1→6)-半乳糖侧链减少到一个或两个半乳糖残基。这些结果表明,AGP 的碳水化合物侧链是由粗糙脉孢菌分泌的 NcAraf1 和 Nc6GAL 的协同作用降解的。