Protein glycosylation and its significance in fungi

为了阐明蛋白质糖化在蛋白质分泌和酶功能表达中的意义，以Taka-淀粉酶A为模型酶，对丝状真菌的糖基化机理进行了分子生物学和生化研究。我们在过去两年中在这两个地点开展的联合工作总结如下：1)携带农杆菌Taka-淀粉酶A基因(AnTAA)的农杆菌产生的AoTAA和AnTAA分别从市售酶制剂和转化子的培养液中得到高度纯化。2)用凝集素印迹分析了AoTAA和AnTAA的糖结构。这两种酶都含有甘露糖作为甘露糖的一种成分。然而，在此分析过程中，在这些制剂中发现了一种高度糖基化的低分子蛋白质。这种低分子质量的糖蛋白干扰了进一步的单糖分析。虽然我们通过多种纯化方法对其进行了纯化，但仍未成功去除酶中的污染物。3)利用荧光标记技术对糖链结构进行了分析，发现AoTAA和AnTAA分别由7和S单糖组成。这表明米曲霉和多年生曲霉的糖基化过程不同。

In order to elucidate the significance of protein glycosylation during protein secretion and in expression of enzymatic function, the glycosylation mechanism in filamentous fungi has been investigated molecular-biologically and biochemically using Taka-amylase A as a model enzyme. Our joint works carried out at both sites during last two years are summarized as follows ;1) The A.oiyzae Taka-amylase A (AoTAA) and Taka-amylase A produced by A.nidulans carrying the A.ozyzae Taka-amylase A gene (AnTAA) were highly purified to homogeneity from commercially available enzyme preparation and the culture broth of an A.nidulans trasformant, respectively.2) The glycan structures of AoTAA and AnTAA were analyzed by lectin blotting. Both enzymes contained mannose as a component of gylcans. However, a highly glycosylated protein with a low molecular mass was found to be contaminated in those preparations during this analysis. This low-molecular-mass glycoprotein interfered further monosaccharide analysis of the glycans. Although we made our best to get rid of this contaminant from AnTAA preparations using several purification methods, we have not yet succeed to remove the contaminant from the enzymes.3) The glycan structures were analyzed by fluorescent labeling technique and we found that AoTAA and AnTAA comprised 7 and S monosaccharides, respectively. This indicates the glycosylation process is different in A.oryzae and A.nidulans