Protein O-mannosyltransferase A of Aspergillus awamori is involved in O-mannosylation of glucoamylase I

Abstract

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...

O -Mannosyltransferase 1 in Aspergillus fumigatus (AfPmt1p) Is Crucial for Cell Wall Integrity and Conidium Morphology, Especially at an Elevated Temperature

“…Evidence has shown that the PMT family is crucial for viability, cell wall integrity, and morphogenesis in several fungal species, such as S. cerevisiae, S. pombe, C. albicans, and C. neoformans (11,27,33,42). Recently, pmtA, encoding an O-mannosyltransferase of the PMT2 subfamily, was shown to be required for the formation of a normal cell wall in Aspergillus species (25,26). It is assumed that the PMT family is also crucial for the growth and, therefore, virulence of A. fumigatus, as seen for C. albicans and C. neoformans.…”

“…Although the structure of O-linked oligosaccharides present in the cell wall peptidogalactomannan of A. fumigatus has been characterized (20), little is known about the genes responsible for the initiation of O-mannosylation assembly. As a matter of fact, only the pmtA gene, which encodes a member of the PMT2 subfamily, has been studied so far, in Aspergillus nidulans and Aspergillus awamori (25,26). Here we report the functional analysis of Afpmt1 of A. fumigatus.…”

Protein O -Mannosyltransferases B and C Support Hyphal Development and Differentiation in Aspergillus nidulans

“…Accordingly, all three pmt genes of A. nidulans contribute to normal hyphal development and are expressed throughout growth, implying that protein O mannosylation plays important roles for this fungal strain. We previously characterized AnpmtA and AapmtA genes encoding PmtA belonging to the PMT2 subfamily (26,27). Here we characterized two Anpmt genes encoding AnPmtB and AnPmtC belonging to the PMT1 and PMT4 subfamilies, respectively.…”

“…Culture filtrates of the transformants were concentrated by centrifugation through Microcon YM-10 (Millipore) filter units and loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detection of GAI was done by immunoblotting using an anti-GAI-antibody as described previously (27).…”

Aspergillus nidulans Protein O -Mannosyltransferases Play Roles in Cell Wall Integrity and Developmental Patterning

“…At least 23 target proteins have been described in yeasts (15). Only three Pmt target proteins have been described in filamentous fungi (12,23,37).…”