Protein glycosylation and its significance in fungi

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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