Protein glycosylation and its significance in fungi

为了阐明蛋白糖基化在蛋白分泌和酶功能表达中的重要意义，本文以taka -淀粉酶A为模型酶，从分子生物学和生化角度研究了丝状真菌糖基化的机制。在过去两年中，我们在两个地点进行的联合工作总结如下：1)分别从市售酵素制剂和假单胞菌转化培养液中纯化出含有假单胞菌taka -淀粉酶A （AoTAA）和携带假单胞菌taka -淀粉酶A基因的假单胞菌产生的taka -淀粉酶A （anttaa）。2)采用凝集素印迹法分析AoTAA和anttaa的多糖结构。这两种酶都含有甘露糖作为多糖的组成部分。然而，在分析过程中，发现了一种低分子质量的高糖基化蛋白在这些制剂中受到污染。这种低分子质量的糖蛋白干扰了对多糖的进一步单糖分析。虽然我们使用了几种纯化方法，尽力从AnTAA制剂中去除这种污染物，但我们还没有成功地从酶中去除这种污染物。3)通过荧光标记技术对其糖结构进行分析，发现AoTAA和anttaa分别由7和S单糖组成。这表明a.m oryzae和A.nidulans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