Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans

Takuji Oka, Tetsu Hamaguchi, Yuka Sameshima, Masatoshi Goto and Kensuke Furukawa

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan

Correspondence
Kensuke Furukawa
kfurukaw{at}agr.kyushu-u.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.


Abbreviations: Dol-P, dolichol phosphate; GAI, glucoamylase I; MNT, mannosyltransferase; PMT, protein O-D-mannosyltransferase

The GenBank accession number for the sequence reported in this paper is AF225551.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Protein glycosylation is a major post-translational modification. Glycosylated proteins play essential roles in eukaryotic cells from fungi to humans (Gemmill & Trimble, 1999; Burda & Aebi, 1999; Strahl-Bolsinger et al., 1999; Lussier et al., 1999). Compared with N-linked oligosaccharides in glycoproteins, which share a relatively common structure, regardless of origin, O-linked oligosaccharides in glycoproteins are diverse, among eukaryotic organisms, with respect to their sugar components and the mode of sugar linkage. O-Glycosylation has been extensively studied in a budding yeast, Saccharomyces cerevisiae (Strahl-Bolsinger et al., 1999; Lussier et al., 1999; Haselbeck & Tanner, 1982; Hausler et al., 1992). The initial reaction of mannose transfer to serine and threonine residues is catalysed by protein O-D-mannosyltransferase (PMT; EC 2.4.1.109) in the endoplasmic reticulum, where dolichol phosphate (Dol-P)-Man is required as an immediate sugar donor (Strahl-Bolsinger et al., 1993). Subsequent extension of mannose moieties is catalysed by the mannosyltransferases (MNTs) {alpha}-1,2-MNT and {alpha}-1,3-MNT, using GDP-Man, in the Golgi (Lussier et al., 1999; Hausler et al., 1992; Romero et al., 1999). Consequently, S. cerevisiae proteins are glycosylated with mannose that is linearly linked to five residues (Strahl-Bolsinger et al., 1999). Seven PMT-encoding genes (PMT1–7), three {alpha}-1,2-MNT-encoding genes (MNT1/KRE2, KTR1, KTR3) and three {alpha}-1,3-MNT-encoding genes (MNN1, MNT2, MNT3), are known to be involved in the complete pathway of O-mannosylation in S. cerevisiae (Gentzsch & Tanner, 1996; Hausler et al., 1992; Lussier et al., 1997, 1999; Romero et al., 1999; Yip et al., 1994). Disruption of three different types of PMT gene resulted in death of the yeast cells (Gentzsch & Tanner, 1996). In addition, some information on O-glycosylation can be obtained from the human pathogenic fungus Candida albicans, which shows yeast–hyphal dimorphism. The pmt1 and pmt6 genes of C. albicans have been characterized to be closely associated with pathogenicity and the transition from round yeast cells into the hyphal form. (Timpel et al., 1998, 2000).

Aspergillus species belong to the filamentous fungi; they include many industrially useable strains such as Aspergillus oryzae, Aspergillus awamori and Aspergillus niger. These fungi produce large amounts of extracellular hydrolases and organic acids, and thus have been used for the production of fermented foods (van den Hondel et al., 1992). Besides these useful Aspergillus strains, opportunistic pathogens such as Aspergillus fumigatus, aflatoxin B1-productive Aspergillus flavus and Aspergillus parasiticus are also known. Therefore, Aspergillus species have been widely investigated in the industrial and medical fields. However, little is known about O-glycosylation in Aspergillus species, although it has been suggested that it is closely associated with secretion (Zakrzewska et al., 2003), stability (Harty et al., 2001; Goto et al., 1999) and localization of proteins (Bourdineaud et al., 1998). Structural analyses of O-linked oligosaccharides of Aspergillus glycoproteins have revealed that O-linked oligosaccharides are predominantly composed of mannose moieties ranging in size from one to three mannose units with linkages of {alpha}-1,2, {alpha}-1,3 and {alpha}-1,6, including not only a linear form but also a branched structure (Pazur et al., 1980; Gunnarsson et al., 1984). Moreover, the O-linked oligosaccharides contain glucose and galactose moieties, in addition to mannose moieties, in the same protein (Gunnarsson et al., 1984; Wallis et al., 1999). Thus, O-linked oligosaccharides in proteins from Aspergillus species are different from those of S. cerevisiae. Recently, Shaw & Momany reported that the swoA mutation, which causes abnormal hyphae development, is identical to the pmtA mutation in Aspergillus nidulans (Momany et al., 1999; Shaw & Momany, 2002). In the present paper, we demonstrate the principal functions of the pmtA gene, and also discuss its role, by providing instances of abnormal cell phenotypes in a pmtA disruptant.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Micro-organisms and growth conditions.
A. nidulans strains FGSC26 (veA+; biA1), FGSC89 (veA+; biA1; argB2) were provided by the Fungal Genetics Stock Center (University of Kansas Medical Center, Kansas City). A. nidulans strains were grown on YG medium (0·5 %, w/v, yeast extract, 1 %, w/v, glucose) or MM medium (1 %, w/v, glucose, 0·6 %, w/v, NaNO3, 0·052 %, w/v, KCl, 0·052 %, w/v, MgSO4.7H2O, 0·152 %, w/v, KH2PO4 and Hunter's trace elements, pH 6·5), as described by Barratt et al. (1965). Liquid growth experiments to allow hyphal development in a submerged culture were done by inoculation of 2x108 conidia into 100 ml YG or MM medium in 500 ml culture flasks. The flasks were shaken at 120 r.p.m. at 30 °C. Standard transformation procedures for A. nidulans were used (Yelton et al., 1984). Plasmids were amplified in Escherichia coli JM109 or XL-1 Blue.

Isolation of the pmtA gene.
Two types of partial genomic DNA library of A. nidulans FGSC26 were constructed. Genomic DNA was digested with SalI and EcoRI, and 3·8 kb DNA fragments were obtained. Genomic DNA was also digested with SalI and ClaI, and 3·7 kb DNA fragments were obtained. The DNA fragments thus obtained were inserted into the corresponding sites of pBluescript-II KS+. E. coli XL-1 Blue transformants carrying two types of genomic DNA library were analysed by colony hybridization. A partial region of the pmtA gene was amplified by PCR with primers pmtA-F (5'-CAGGCCGTAACCTTCATTCCCA-3') and pmtA-R (5'-CCGGCGCGTAGATAACAACCC-3') and was used as a hybridization probe. Two positive clones carrying pBS-C-pmtA and pBS-N-pmtA were isolated from the two types of genomic library. The 1·86 kb SalI–SphI fragment from pBS-N-pmtA and the 2·69 kb SphI–BamHI fragment from pBS-C-pmtA were co-inserted into the SalI and BamHI sites of pBluescript-II KS+, to yield pBS-pmtA, which carries a 4·55 kb SalI–BamHI fragment containing the entire pmtA gene. In order to isolate the cDNA of pmtA, RT-PCR was performed with a High Fidelity RNA PCR kit (Takara), using the primers pmtA-RT-F (5'-TTGGATCCGAAATGGCTGAAATTGGCTTTG-3') and pmtA-RT-R (5'-GTTCCTCGAGGTTAGCGATTCGCCAAC-3').

Analysis of pmtA transcription.
Total RNA was isolated by Trizol reagent (Gibco-BRL). An 18 µg sample of total RNA was separated by electrophoresis in a 1·0 % agarose gel with 15 % (v/v) formaldehyde and transferred to a Hybond-N membrane (Amersham Biosciences). Hybridization with DIG-labelled probes (Roche) and subsequent detection with CDP-star (Roche) were done according to the supplier's manual. Primer-extension experiments using the mRNA of strain FGSC26 were done with the Primer Extension System-AMV Reverse Transcriptase (Promega) and the primer PE-pmtA (5'-IRD800-GGTCGATGCAAAGCCAATTTCAGCCATG-3'). A sequencing reaction was performed using a Thermo Sequenase Cycle Sequencing Kit (USB) with a 1·5 µM ddNTP (Amersham Biosciences) and 150 µM dNTP (Promega) mixture. Analysis of reaction products was done on a model LIC4200L-2G DNA sequencer (LI-COR).

Construction of the pmtA disruptant.
A plasmid, pBS-{Delta}pmtA : : argB, for disruption of the pmtA gene, was constructed as follows. The 1·7 kb EcoRI fragment of pDC1 (Aramayo et al., 1989), containing the argB gene of A. nidulans, was inserted into the middle of the pmtA gene at the EcoRI site of pBS-pmtA. The 6·0 kb DNA fragment containing {Delta}pmtA : : argB that had been prepared by digestion of pBS-{Delta}pmtA : : argB with BssHII was used for transformation of A. nidulans FGSC89. The disruption of the pmtA gene in the argB+ transformants was confirmed by Southern blot analysis. The 0·9 kb terminator region of pmtA was amplified by PCR with the primers pmtA-TF (5'-AAAAGGGCCCGAACCGTGATAGAGCG-3') and pmtA-TR (5'-AAAAGGGCCCGGATCCCGCATTGTCC-3') and used as a probe.

Protein O-mannosylation activity in vitro.
A. nidulans was grown on MM medium for 24 h at 30 °C. Dol-P-[14C]Man was synthesized according to the methods of Sharma et al. (1974). The in vitro peptide assay for O-mannosylation activity was performed according to Gentzsch & Tanner (1996). Dol-P-[14C]Man as a sugar donor and Ac-YATAV-NH2 as an acceptor peptide were used as substrates. The reaction mixture contained 5000 c.p.m. Dol-P-[14C]Man, 1·5 mM acceptor peptide, 7 mM Tris/HCl, pH 7·5, 7 mM MgCl2, 0·14 % (w/v) Triton X-100 and 0·2–0·3 mg ml–1 microsomal membranes of A. nidulans. The mixtures were incubated at 30 °C for 20 min. The amount of [14C]mannosylated peptide was measured by a liquid scintillation counter. The assay was done three times independently.

Expression of pmtA and glaA.
The plasmids for expression of pmtA and the glucoamylase-encoding glaA were constructed as follows. The 4·55 kb SalI–BamHI fragment carrying the pmtA from pBS-pmtA was inserted into the corresponding sites of pUC19, to yield pUC-pmtA. pUC-pmtA was digested by PstI and SmaI, and the resultant pmtA fragment was inserted into the PstI and SmaI sites of pPTR-I, carrying the ptrA gene as a selection marker (Takara), to yield pPTR-pmtA. The glaA gene of A. awamori, from pBR-glaA, (Goto et al., 1997) was inserted into the SmaI site of pPTR-I, to generate pPTR-glaA. The pPTR-pmtA and pPTR-glaA were linearized by digestion with PstI and HindIII, respectively. Transformants showing pyrithiamine resistance were selected on MM medium supplemented with 0·1 µg pyrithiamine ml–1 and 0·8 M NaCl. Transformants carrying the glaA gene were cultured in 100 ml MM medium with 1 % (w/v) maltose, to induce glaA expression, and with 20 mM tunicamycin as an inhibitor for N-glycosylation of protein, for 24 h at 30 °C.

Colony growth rate.
Colony growth rates were measured as described by Kellner & Adams (2002). Briefly, conidia from each of the strains were point-inoculated into the centre of agar medium plates and incubated at 37 °C. Colony diameters were measured at 29, 49, 66 and 91 h. The growth rates were observed for each colony in mm h–1 for each of the above three intervals, and the values were averaged across the entire time interval. The growth rates were expressed as a percentage of the growth rate of the wild-type (FGSC26) on YG medium, which was 0·59 (±0·085) mm h–1 (n=15). Measurements of independent growth rates for all strains were done 15 times.

Analysis of efficiency of conidiation.
Efficiency of conidiation was analysed as described by Motoyama et al. (1997). Briefly, approximately 105 conidia were spread onto an 84 mm MM medium plate. After 5 days of incubation at 30 °C, the conidia were suspended in 5 ml 0·01 % (w/v) Tween 20 solution and counted using a haemocytometer.

Microscopy.
The germlings of A. nidulans were observed according to the protocol described by Harris et al. (1994). Conidia were inoculated into YG liquid medium, and the culture was poured into a Petri dish containing a glass coverslip. After incubation at 30 °C for 6–14 h, the germlings adhering to the coverslip were fixed in 3·7 % (v/v) formaldehyde, 50 mM phosphate buffer (pH 7·0) and 0·2 % (w/v) Triton X-100 for 30 min. Coverslips were then washed with water and incubated with 10 ng ml–1 of fluorescent brightener 28 (Calcofluor white; Sigma) for 5 min. The coverslips were washed again and mounted on a slide glass. Germlings were observed using the Eclipse E600 (Nikon).

Preparation of microsomal membrane and cell-wall fractions.
A. nidulans was grown on MM or YG medium for 24 h, harvested, washed with ice-cold TM buffer (50 mM Tris/HCl, pH 7·5, 0·3 mM MgCl2) three times and broken twice in a French press (Ohtake) in the same buffer. The lysed cells were centrifuged at 3000 g for 20 min. The resultant pellet was washed ten times with 250 mM phosphate buffer (pH 7·0), washed with distilled water five times, lyophilized, and designated a cell-wall fraction. The supernatant was centrifuged at 100 000 g for 90 min, suspended in TM buffer, and designated a membrane fraction.

Analysis of the contents of glucan and chitin.
Analysis of the glucan and chitin contents was done by the methods described by Borgia & Dodge (1992), with a slight modification. Cells were cultured in YG medium for 24 h at 30 °C. For the quantification of total glucan, 10 mg of cell walls was suspended in 1·0 ml 98 % formic acid, heated at 100 °C for 20 min and centrifuged. The supernatant was dried in a vacuum desiccator, and the amount of neutral carbohydrate was estimated by the phenol-sulfuric acid method (Dubois et al., 1956). For the quantification of alkali-soluble and alkali-insoluble fractions, 10 mg of cell walls was suspended in 1·0 ml distilled water, heated at 100 °C for 30 min and centrifuged. The pellet was suspended in 1·0 ml 1·0 M KOH solution and heated at 70 °C for 30 min prior to centrifugation. The supernatant was dried and used to estimate the amount of alkali-soluble glucan, as above. The alkali-insoluble pellet was suspended in 1·0 ml 98 % formic acid and heated at 100 °C for 20 min, dried and used to estimate the amount of alkali-insoluble glucan. For the quantification of total GlcNAc, 10 mg of cell walls was digested with 2 mg Yatalase ml–1 (Takara) in 250 mM phosphate buffer (pH 7·0) for 16 h. After digestion, the suspension was centrifuged, dried and used to estimate the amount of total GlcNAc by the Morgan–Elson method (Reissig et al., 1955). The analyses were done three times independently.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of the pmtA gene
The expressed sequence tags of A. nidulans with homology to the amino acid sequences of S. cerevisiae Pmt1-4p (Gentzsch & Tanner, 1996) were identified in a clone of h4a11a1.r1 by a database search using the TBLASTN algorithm (Altschul et al., 1997) against dbEST (Expressed Sequence Tag database). The resultant nucleotide sequence was used as a probe to isolate the pmtA gene from the genomic DNA library of A. nidulans FGSC26. The pmtA cDNA was cloned by amplification with RT-PCR. The nucleotide sequence and deduced amino acid sequence were deposited in GenBank (accession no. AF225551).

Transcription of the pmtA gene
Northern blot analysis of pmtA was carried out at various growth stages, using the pmtA cDNA as a probe. The amounts of the pmtA transcripts under incubation in MM medium were almost comparable with the {gamma}-actin gene from 16 h to 48 h (Fig. 1a). This result indicates that pmtA is transcribed throughout hyphal growth. The transcriptional start points of the pmtA gene were determined by primer-extension analysis, and indicated that pmtA has at least five transcriptional initiation sites, at –27, –46, –75, –127 and –226 bp upstream from the translational start site (Fig. 1b). Analysis of the 5'-noncoding region revealed the presence of putative promoter-proximal elements of a TATA-like sequence involved in the expression of the pmtA gene. The promoter contains a putative binding sequence of the pH signalling transcription factor PacC (Espeso et al., 1997) 373 bp upstream from the translation start codon (Fig. 1b).



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Fig. 1. Transcriptional analysis of pmtA of A. nidulans. (a) Transcription of pmtA. Total mRNA of A. nidulans wild-type strain FGSC26 was prepared after growth in MM medium at 30 °C. Each lane was loaded with 18 µg RNA. Northern blot hybridization was performed using pmtA and the {gamma}-actin gene (accession no. M22869) as probes, and total RNA was stained with ethidium bromide (EtBr). (b) Nucleotide sequence and primer extension analysis of the 5' upstream region of the pmtA gene of A. nidulans. TATA boxes are underlined. Transcriptional initiation positions are shown by arrows. The PacC-protein-binding consensus sequence is double-underlined (Espeso et al., 1997). Numbering starts from the translation initiation site.

 
Disruption of the pmtA gene
To examine whether the pmtA gene product functions as a protein O-D-mannosyltransferase, we constructed a pmtA null mutant. For this purpose, the 6·0 kb BssHII DNA fragment carrying {Delta}pmtA : : argB was introduced into the A. nidulans FGSC89 strain (Fig. 2a). Isolated genomic DNA from the selected argB+ transformants was analysed by Southern blotting, using the 0·9 kb terminator region of the pmtA gene as a probe (Fig. 2b). Southern blot analysis revealed that site-specific recombination occurred at the pmtA region, and that a single copy of the 6·0 kb fragment was integrated into the chromosomal DNA. Two isogenic pmtA-disruptant strains, P3 and P4, were isolated from approximately 150 argB+ transformants, and strain P3 was used for further analysis.



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Fig. 2. Disruption of A. nidulans pmtA. (a) Schematic representation of the pmtA disruption. Regions used as probes for Southern blot analysis are shown as bars. Abbreviations: H, HincII; B, BamHI; E, EcoRI. (b) Southern blot analysis of HincII-digested total DNA. Lane 1, strain P3; lane 2, wild-type strain FGSC89. (c). Protein O-D-mannosyltransferase activity in vivo. Proteins in the culture filtrate of strain P3+pPTR-glaA (lanes 1 and 3) and wild-type strain FGSC89+pPTR-glaA (lanes 2 and 4) were separated on 7 % SDS-PAGE, and GAI was detected by immunoblotting using anti-GAI antiserum. Tunicamycin was added as an inhibitor of N-glycosylation (lanes 1 and 2). Precision Plus protein standards (Bio-Rad) were used as a molecular size marker (lane 5). The molecular mass of GAI was estimated by an image analyser (Bio-profile V6.0) after SDS-PAGE.

 
We tested membrane proteins extracted from the wild-type strain and the pmtA disruptant for their ability to synthesize O-mannosylated peptides, using Dol-P-[14C]Man as a sugar donor. Membrane proteins from the wild-type strain exhibited a high level of mannosylation activity (8521±503 c.p.m. mg–1 h–1) toward the Ac-YATAV-NH2 peptide, while the activity of pmtA disruptant P3 (523±179 c.p.m. mg–1 h–1) was as low as 6 % of that of the wild-type. The result provided evidence that the pmtA gene encodes a functional protein O-D-mannosyltransferase. We then introduced the glaA gene of A. awamori into the pmtA disruptant and the wild-type strain in order to confirm the in vivo O-mannosylation activity encoded by pmtA. Glucoamylase I (GAI), encoded by glaA, is an extracellular protein consisting of three domains. These are the amino-terminal catalytic domain covering amino acids 1–469, the Ser/The-rich region including 470-ATGGTTTTATTTGSGGVTSTSKTTTTASKTSTTTSSTS-507, and the carboxy-terminal starch-binding domain of amino acids 508–615 (Hayashida et al., 1989). The SDS-PAGE mobility of GAI secreted from the pmtA disruptant was greater than that of the wild-type strain (Fig. 2c). No significant effects of tunicamycin were seen on the mobility of GAI on SDS-PAGE between the wild-type and the pmtA disruptant under the experimental conditions used (Fig. 2c). The molecular masses of GAI from the wild-type strain and the pmtA disruptant were estimated to be 70 kDa and 68 kDa, respectively. It is known that the glucoamylase of A. awamori has a variety of sizes, due to the heterogeneity of the attached oligosaccharides (Sauer et al., 2000). It is therefore reasonable that the immunoblots showed a population of glucoamylase molecules with a variety of sizes, spanning approximately a 5 kDa range. This result indicates that the PmtA is functional for a foreign secretory protein in vivo.

The pmtA disruptant formed a colony approximately 90 % of the diameter of that of the wild-type strain, when cultured on MM medium at 30 °C for 3 days (Fig. 4). However, in the case of growth on YG medium at 37 °C, the pmtA disruptant P3 showed remarkable inferiority in its hyphal extension ability: its growth rate was reduced to 41·8 % compared to the wild-type (Table 1). We attempted to obtain transformants whose pmtA mutation was complemented, where one to three copies of the pmtA gene were integrated via introduced plasmid pPTR-pmtA. The colony growth rate of the transformants (strain P3 carrying wild-type pmtA) was fully restored (Table 1), indicating that the abnormal hyphal extension is due to the pmtA mutation. The growth rate was fully restored on YG medium plates in the presence of 0·6 M KCl. The growth-rate recovery was also observed at the same level when 0·8 M NaCl and 1·2 M sorbitol were used, instead of 0·6 M KCl, as an osmotic stabilizer (data not shown). In addition, we often observed lysis of the hyphal tip in the MM liquid medium without the stabilizer. However, the hyphal lysis was repressed when the stabilizer was incorporated. Morphology of the germlings in the early growth stage was observed by microscopy. On YG and MM media, swollen hyphal formation (balloon formation) appeared at the apical or umbilical regions of the hyphae (Fig. 3a). This morphology was observed in moribund regions, in particular.



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Fig. 4. Increased sensitivity to antifungal reagents displayed by A. nidulans pmtA disruptant P3 compared to that of wild-type strain FGSC26. Strains were grown in the presence of Congo red (CR; 450 µg ml–1) or Calcofluor white (CW; 300 µg ml–1) on MM medium for 3 days at 30 °C. MM, control.

 

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Table 1. Growth rate of A. nidulans wild-type strain FGSC26 and pmtA mutant strain P3

Values in parentheses show the growth rate as a percentage of that of the wild-type strain. Growth rate values are in mm h–1.

 


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Fig. 3. Hyphae structure of A. nidulans pmtA disruptant P3 (a) and wild-type strain FGSC26 (b) was observed as described in Methods. Right panels, fluorescent image of the cell stained with Calcofluor white. Left panels, phase-contrast image.

 
Effects of the pmtA disruption on conidia formation
We measured the conidiation efficiency of the pmtA disruptant and the wild-type strain. Conidiation was repressed in the pmtA disruptant, in comparison with the wild-type strain, notably on MM medium. The formation of conidia in the pmtA-disrupted strain P3 was reduced by approximately 20 % compared with that of the wild-type strain.

pmtA disruption causes hypersensitivity to antifungal reagents
Congo red and Calcofluor white are known to be adsorbed on cell walls composed of polysaccharides, and therefore exhibit antifungal effects. In the presence of Congo red and Calcofluor white, the hyphal growth of the pmtA disruptant on MM medium was markedly inhibited compared with the wild-type strain (Fig. 4).

Cell-wall chitin and {beta}-glucan content of wild-type and pmtA-disruptant strains
The alkali-soluble fraction contains {alpha}-glucan and soluble {beta}-1,3-/1,6-glucan. The alkali-insoluble fraction contains {beta}-1,3-/1,6-glucan covalently linked to chitin (Borgia & Dodge, 1992; Fontaine et al., 2002; Lee et al., 2002). The pmtA disruptant showed a 20 % decrease in the alkali-insoluble fraction. The alkali-soluble fraction and the GlcNAc content of the cell walls of the pmtA disruptant were increased by 44 % and 33 %, respectively, compared with the wild-type strain. The total sugar content in the cell walls of the pmtA disruptant was comparable to that of the wild-type strain (Table 2). The alkali-insoluble fraction is believed to be responsible for fungal cell-wall rigidity (Fontaine et al., 2002). The decrease in the alkali-insoluble fraction indicates that the cell wall was weakened.


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Table 2. Cell-wall composition of A. nidulans wild-type strain FGSC26 and pmtA mutant strain P3

The amount of total sugar is expressed as the amount of glucose equivalent. Values in parentheses are percentages based on the wild-type strain.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Protein O-glycosylation is known as an important post-translational modification of proteins in secretory traffic. Among a variety of O-glycosylations, protein O-mannosylation occurs in Aspergillus. Reactions catalysed by protein O-D-mannosyltransferase play a key role in the O-mannosylation of proteins because they are involved in the initial O-mannosylation reaction. Several Pmt proteins have been identified in S. cerevisiae, C. albicans, Drosophila melanogaster and man (Gentzsch & Tanner, 1996; Timpel et al., 1998, 2000; Martin-Blanco & Garcia-Bellido, 1996; Willer et al., 2002). However, little is known about the character of the Pmt in Aspergillus. In the present paper, we constructed a pmtA disruptant and analysed the properties of the cell wall.

The pmtA disruption gave a phenotype of A. nidulans identical to the temperature-sensitive swoA mutant reported by Momany et al. (1999) and Shaw & Momany (2002). Inhibition of hyphal extension, multinucleation of the cell, and enhanced susceptibility to heat and Calcofluor white were observed. In addition to the properties of the swoA mutant, we observed hypersensitivity to Congo red (Fig. 4) and a decrease in conidia formation. Disruption of the pmtA gene revealed that it encodes the functional protein O-D-mannosyltransferase. The pmtA-disrupted strain exhibited greatly reduced protein O-D-mannosyltransferase activity (6 %), compared to the wild-type. The remnant activity would be derived from Pmt isozymes. Thus, A. nidulans is assumed to possess several pmt genes, as in other organisms (Gentzsch & Tanner, 1996; Timpel et al., 2000). In fact, we cloned two genes homologous to pmtA and confirmed their transcripts in A. nidulans (unpublished data).

In order to confirm in vivo mannosylation activity toward a secretory protein, GAI of A. awamori was heterologously expressed in A. nidulans. The Ser/Thr-rich region of GAI, which could be the target for O-glycosylation, contains 27 hydroxyamino acids among 38 amino-acid residues (Hayashida et al., 1989). Unexpectedly, only a 2 kDa difference in the molecular mass of GAI was observed between the wild-type strain and the pmtA disruptant, suggesting that approximately 11 mannose moieties are absent in the pmtA disruptant. This result supports the fact that PmtA protein catalyses O-mannosylation with an inherent substrate specificity for certain proteins. Gentzsch & Tanner reported that, in S. cerevisiae, Pmt proteins have distinct substrate specificities: for proteins that contain Ser or Thr (Gentzsch & Tanner, 1997). Therefore, the slight difference in glycosylation in GAI would be due to a lower preference of A. nidulans PmtA for the foreign GAI polypeptide as the substrate.

It is well documented that some triple mutants of PMT1–4 show a lethal phenotype in S. cerevisiae. On the other hand, all single and double mutants of PMT were viable (Gentzsch & Tanner, 1996). The pmtA disruptant of A. nidulans is also viable. However, a defect in the PmtA function resulted in repression of normal hyphal growth and normal conidial development, particularly under conditions of low osmotic potential. pmtA is transcribed at abundant levels through the early to late growth-stages in the wild-type strain (Fig. 1a). Because hyphal extension is in concert with cell-wall formation in filamentous fungi, the pmtA transcribed abundantly in conventional growth would be required for cell-wall formation. The pmtA disruptant of A. nidulans showed higher sensitivity to antifungal agents than the wild-type strain (Fig. 4). A similar response was observed in C. albicans pmt1 and pmt1pmt6 mutants (Timpel et al., 2000). Therefore, it is not surprising that the same effects are observed in the pmtA disruptant of A. nidulans. The addition of osmotic stabilizers to the growth medium resulted in recovery of the defect in hyphal growth of the pmtA disruptant. These results indicated that the structure and mechanical rigidity of the cell walls are drastically altered by the pmtA disruption. Unlike the pmt mutants of C. albicans, the pmtA disruption also caused swollen hyphae locally in the mycelia, although, overall, mycelial development did take place (Fig. 3a). In Aspergillus species, the formation of swollen structures in the hyphae was reported in mutants of chitin synthase or NAD-dependent glycerol-3-phosphate dehydrogenase (Borgia et al., 1996; Horiuchi et al., 1999; Fillinger et al., 2001). The cell-wall rigidity of those mutants was defective because the biosynthesis of chitin or glycerol was significantly reduced. These morphological rigidity defects were restored by the addition of an osmotic stabilizer.

Cell-wall components are functionally classified into two groups: the skeletal polysaccharides and the polysaccharides of the wall matrix (Farkas, 1985). The skeletal polysaccharides include {beta}-1,3-glucan interconnected covalently with {beta}-1,6-glucan and chitin, which are highly crystalline and provide mechanical rigidity to the wall. These polysaccharides were recovered as an alkali-insoluble fraction (Borgia & Dodge, 1992; Fontaine et al., 2002; Lee et al., 2002). The alkali-soluble fraction contains {alpha}-glucan and soluble {beta}-1,3/1,6-glucan, which fills the space between the skeletal polysaccharides and serves as a cementing substance (Borgia & Dodge, 1992; Farkas, 1985). The pmtA disruptant exhibited a significant alteration in the carbohydrate composition of the cell-wall fractions (Table 2). The cell walls in the pmtA disruptant changed to a defective structure, due to the reduction of the skeletal polysaccharides of the {beta}-glucans and the increase in chitin content. The polysaccharides of the wall matrix increased in the pmtA disruptant. Therefore, the pmtA disruptant gives rise to critical damage to the skeletal structure and wall matrix of the cell wall. Accordingly, mycelial extension can be repressed in the pmtA disruptant.

In S. cerevisiae, it is known that the KRE1 and KRE9 genes encode O-glycosylated proteins involved in the synthesis of {beta}-1,6-glucan. KRE1 and KRE9 mutants exhibit fragile cell-wall phenotypes, due to the reduction of the amount of {beta}-1,6-glucan in the cell wall (Brown et al., 1993; Brown & Bussey, 1993). Therefore, the underglycosylation caused by the pmtA disruption may affect the synthesis of {beta}-1,6-glucan. It has also been reported that disruption of the {beta}-glucan synthase gene results in an increase in cell-wall chitin content in S. cerevisiae. (Garcia-Rodriguez et al., 2000). The increase in chitin content in the pmtA disruptant of A. nidulans may be due to a similar mechanism, through a kind of salvage mechanism for covering cell-wall weakening. Philip & Levin (2001) reported that Wsc1 and Mid2, which are cell-surface sensors responsible for cell-wall integrity through the MAP kinase pathway, are modified by ScPmt2p. Thus, one cannot exclude the possibility, in A. nidulans, that the impairment in O-mannosylation triggers the cascade of events that leads first to damage to cell-wall integrity and then to the cell response involving its repair through cell-wall compensatory mechanisms. Alternatively, PmtA could be directly involved in the mannosylation of proteins covalently attached to the polysaccharides of the wall matrix, whereby the pmtA disruption may cause alteration of the cell-wall structure.

A number of proteins secreted into the culture and localized in the cell wall are O-glycosylated (Mrsa & Tanner, 1999; Woo et al., 2003). However, in Aspergillus, most of the O-glycosylated proteins localized in the cell walls or plasma membrane have not yet been identified. We are currently involved in an investigation using the pmtA disruptant to identify the target proteins that PmtA modulates in structure and function.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 24 December 2003; revised 5 February 2004; accepted 1 March 2004.



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