Protein O-glycosylation is essential for protein modification and plays important roles in eukaryotic cells. O-Mannosylation of proteins occurs in the filamentous fungus Aspergillus. The structure and function of the pmtA gene, encoding protein O-D-mannosyltransferase, which is responsible for the initial O-mannosylation reaction in Aspergillus nidulans, was characterized. Disruption of the pmtA gene resulted in the reduction of in vitro protein O-D-mannosyltransferase activity to 6 % of that of the wild-type strain and led to underglycosylation of an extracellular glucoamylase. The pmtA disruptant exhibited abnormal cell morphology and alteration in carbohydrate composition, particularly reduction in the skeletal polysaccharides in the cell wall. The results indicate that PmtA is required for the formation of a normal cell wall in A. nidulans.

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Industrially important extracellular enzymes from filamentous fungi are often O-mannosylated. The structure and function of the pmtA (AapmtA) gene encoding the protein O-D-mannosyltransferase of Aspergillus awamori were characterized. The AapmtA disruptant, designated AaPMTA, was constructed by homologous recombination. The strain AaPMTA exhibited fragile cell morphology with respect to hyphal extension, as well as swollen hyphae formation and conidia formation in potato dextrose medium. Moreover, the AapmtA disruptant showed increased sensitivity to high temperature and Congo red. Thus, the AaPmtA protein is involved in the formation of the normal cell wall. The strain AaPMTA could grow well in liquid synthetic medium and secrete glucoamylase I (GAI-AaPMTA) to a similar extent to the wild-type strain (GAI-WT). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the GAIs revealed that approximately 33 mannose moieties of GAI were absent in strain AaPMTA. This result indicates that the AaPmtA protein is responsible for the transfer of mannose to GAI. Structural analysis of the O-linked oligosaccharides of GAI also demonstrated that the AapmtA disruption resulted in a reduction of the amounts of O-linked oligosaccharides, such as D-mannose and a-1,2-mannotriose, in GAI-AaPMTA. However, the amount of a-1,2-mannobiose was comparable between GAI-WT and GAI-AaPMTA. The result suggests the presence of a compensatory mechanism in the synthetic pathway of O-mannosylation in A. awamori. INTRODUCTIONProtein glycosylation, which is a major post-translational modification, plays essential roles in eukaryotic cells from fungi to mammals (Burda & Aebi, 1999;Gemmill & Trimble, 1999). Compared with N-linked oligosaccharides in glycoproteins, which share a relatively common structure regardless of their origins, O-linked oligosaccharides in glycoproteins are diverse among the eukaryotic organisms with respect to both their sugar components and the mode of sugar linkages (Haltiwanger & Lowe, 2004;Strahl-Bolsinger & Scheinost, 1999;Willer et al., 2003). O-Mannosylation, which is commonly found in the glycoproteins of fungi, has been extensively studied in the budding yeast Saccharomyces cerevisiae (Gentzsch & Tanner, 1996;Lussier et al., 1997; StrahlBolsinger & Scheinost, 1999). The initial reaction of mannose transfer to serine and threonine residues in proteins is catalysed by protein-O-D-mannosyltransferase (Pmt) in the endoplasmic reticulum, where dolichol phosphate (Dol-P)-Man is required as an immediate sugar donor (Gentzsch & Tanner, 1996). Subsequently, O-mannosylation is linearly elongated by up to seven mannose residues in the Golgi complex by mannosyltransferases (Mnts) that utilize GDPmannose as the mannosyl donor for the elongation process. At least six Pmt-encoding genes (PMT1-6), three a-1,2-Mnt-encoding genes (MNT1/KRE2, KTR1, KTR3) and three a-1,3-Mnt-encoding genes (MNN1, MNT2, MNT3) are known to be involved in the complete reactions of O-mannosylation in S. cerevisiae (Lussier...

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