Curr Genet (2003) 43: 11–16 DOI 10.1007/s00294-003-0368-5
R ES E AR C H A RT I C L E
Anna Zakrzewska Æ Andrzej Migdalski Markku Saloheimo Æ Merja E. Penttila Gra_zyna Palamarczyk Æ Joanna S. Kruszewska
cDNA encoding protein O-mannosyltransferase from the filamentous fungus Trichoderma reesei ; functional equivalence to Saccharomyces cerevisiae PMT2 Received: 15 October 2002 / Revised: 9 December 2002 / Accepted: 18 December 2002 / Published online: 13 February 2003 Springer-Verlag 2003
Abstract O-Mannosylation is suggested to be essential for protein secretion in Trichoderma reesei. In protein O-glycosylation, the first mannosyl residue is transferred to a serine or threonine hydroxyl group of the protein from dolichyl phosphate mannose by protein O-mannosyltransferase. In Saccharomyces cerevisiae, seven PMT genes have been cloned coding for these enzymes. In the present work, the characterisation of the pmt1 cDNA from T. reesei is reported. Sequence analysis of the predicted protein revealed the highest similarity to Schizosaccharomyces pombe Pmt and to Pmt4p of Saccharomyces cerevisiae. In contrast, expression of the T. reesei cDNA in various S. cerevisiae pmt mutants showed functional similarity to the yeast Pmt2 protein. Keywords pmt gene Æ Protein glycosylation Æ O-Mannosylation Æ Secretion
Introduction The majority of Trichoderma reesei secretory proteins, including the main secretory enzyme cellobiohydrolase I (CBH I), are O-mannosylated glycoproteins (Palamarczyk et al. 1998). The O-glycosylation process in Trichoderma has been suggested to be essential for protein secretion (Kubicek et al. 1987). This fact initiated intensive studies on O-glycosylation in T. reesei
Communicated by S. Hohmann A. Zakrzewska Æ A. Migdalski Æ G. Palamarczyk J.S. Kruszewska (&) Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin´skiego 5a, 02-106 Warsaw, Poland E-mail:
[email protected] M. Saloheimo Æ M.E. Penttila VTT Biotechnology, P.O. Box 1500, 02044 VTT, Finland
and resulted in the cloning of two genes (mpg1, dpm1) encoding enzymes engaged in O-glycosylation (Kruszewska et al. 1989, 1998, 1999, 2000). The O-mannosylation process is initiated by transfer of a mannosyl residue from dolichyl phosphate mannose (DPM) to the serine/threonine –OH group of a protein. The reaction is catalysed by protein O-mannosyltransferases (Pmt) encoded by the PMT genes. In Saccharomyces cerevisiae, seven PMT genes sharing 50–55% amino acid sequence identity were identified. Involvement in protein O-mannosylation was demonstrated for five of them (PMT1, 2, 3, 4, 6; Gentzsch and Tanner 1996, 1997). In vivo studies demonstrated different substrate specificity for various protein O-mannosyltransferases. Amino acid sequence analysis of the protein substrates and mannosyl transfer activity measurements in various pmt mutants gave no clear explanation for the basis of the observed substrate specificity (Gentzsch and Tanner 1997). Ten O-glycosylated proteins were analysed and six of them (chitinase, Bar1p protease, Pir2p/Hsp150p, a-agglutinin, Kre1p, Kre9p) were glycosylated by Pmt1p and Pmt2p. The other four (Kex2p, Gas1p, Fus1p, Axl2p) showed a decrease or lack of O-glycosylation in a pmt4 disruptant strain. Single pmt deletions gave no changes in strain viability but some triple and double mutants (pmt1pmt2pmt3, pmt2pmt3, pmt2pmt4) are able to growth only when osmotically stabilised (Gentzsch and Tanner 1996), thus demonstrating defects in cell wall integrity. Some triple mutants are not viable (pmt1pmt2pmt4, pmt2pmt3pmt4). Pmtp orthologues have been identified from the yeasts Schizosaccharomyces pombe and the human pathogen Candida albicans (Timpel et al. 1998). The pmt protein from Sch. pombe has not been functionally characterised. Homozygous pmt1 mutants of C. albicans were avirulent to mouse, while the heterozygous PMT1/pmt1 strain gave reduced virulence. A gene with nucleotide sequence similar to yeast PMT genes has also been described from Drosophila melanogaster (Martin-Blanco and Garcia-Bellido 1996). Homozygous Drosophila mutants of that gene
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have strong defects in embryonic muscle development. A gene (POMT1) with sequence similarity to PMTs has been identified in the human genome (Jurado et al. 1999). The latest results from Beltran-Valero De Bernabe et al. (2002) show that mutations in the POMT1 gene give rise to Walker–Warburg syndrome, a disorder characterised by muscular dystrophy and complex brain and eye abnormalities. In the present paper, we identify a new member of the protein O-mannosyl-transferase family from T. reesei and report partial complementation of the pmt2 mutation in S. cerevisiae by the Trichoderma pmt gene.
Materials and methods The S. cerevisiae strains used for complementation tests are specified in Table 1. Escherichia coli DH5a (Bethesda Research Laboratories) was used as a plasmid host. The T. reesei RutC-30 cDNA libraries were described by Sta˚lbrand et al. (1995) and MargollesClark et al. (1996) and were constructed in the Lambda ZAP II vector (Stratagene) and in the yeast expression plasmid pAJ401 (Saloheimo et al. 1994). Yeast strains were grown in SC medium (Sherman 1991) with the necessary supplements. For yeast transformation, the one-step transformation method of Chen et al. (1992) was used.
Isolation of the T. reesei pmt gene cDNA library screening by plaque hybridisation and other standard DNA manipulations were done as described by Sambrook et al. (1989). For probe synthesis, [32P]a-dATP and the Megaprime DNA labelling system (Amersham) were used. Plasmids were isolated as described by Short et al. (1988) and Sambrook et al. (1989). The inserts were sequenced using a PCR sequencing kit (DYEnamic ET terminator cycle sequencing kit, Amersham) and an ABI 373 sequencer (Perkin-Elmer).
Construction of expression plasmids For expression of Trichoderma pmt1 in S. cerevisiae, a cDNA fragment of about 2,400 bp, containing the pmt1 open reading frame, was amplified by PCR and cloned into pNEV (with URA3 marker) and YEP18 (with LEU2 marker) plasmids between the PMA1 (plasma membrane H+ ATPase) promoter and terminator (gifts from Prof. F. Karst, Laboratoire de Genetique de la Levure, Universite de Poitiers, France). Both plasmids were cut with the NotI restriction enzyme, blunted with a DNA polymerase I Klenow fragment and ligated with the pmt1 PCR product. Ligation direction was examined by restriction analysis.
Protein O-mannosyltransferase assay S. cerevisiae strains were cultivated at 30 C in 1 l of SC-Leu or SCUra medium with supplements and 2% glucose as the carbon source up to an optical density at 600 nm of 1.0, after which the cells were harvested by centrifugation and resuspended in 25 ml of 150 mM Tris buffer (pH 7.4) containing 15 mM MgCl2 and 9 mM 2-mercaptoethanol. The cells were homogenised with 0.5-mm glass beads in a bead-beater and the homogenate was centrifuged at 4,000 g for 10 min to remove unbroken cells and cell debris. The supernatant was centrifuged for 1 h at 50,000 g and protein O-mannosyltransferase activity was measured in the pelleted membrane fraction by 2 h of incubation at 30 C with 0.02 lCi DolP-[14C]Man (303 Ci/mol) as a substrate, as described by StrahlBolsinger and Tanner (1991). Total membrane proteins (about 300 lg) were used as the sugar acceptor. The protein O-mannosyltransferase activity was presented as picomoles of [14C]Man incorporated into 1 mg of membrane protein during 2 h.
Immunoblotting Yeast cells were grown to the logarithmic phase, washed with 50 mM Tris-HCl, pH 7.4, 50 mM MgCl2 and broken with glass beads in the same buffer. Cell walls were centrifuged down at 3,000 g and the pellet was resuspended in the same buffer. Cell wall proteins (about 100 lg/lane) from the resulting pellet were separated by SDS-PAGE on a 6% gel. Chitinase was detected by immunostaining with an antibody against the deglycosylated protein (a gift from Prof. Widmar Tanner, Lehrstuhl fur Zellbiologie und Pflanzenphysiologie, Universitat Regensburg, Germany). Plasmalemma-bound gp115/Gas1p was detected with anti Gas1p antibody (kindly provided by Dr. Thomas Aust, Biozentrum, University of Basel, Switzerland). Plasma membranes were obtained as described above.
Results and discussion Sequence analysis of the T. reesei pmt1 cDNA clone (accession number AF526877) The pmt1 gene was first discovered while screening a T. reesei expression library (Margolles-Clark et al. 1996) in yeast. This screening was not connected to protein glycosylation. Sequencing of one cDNA clone obtained in the screening revealed that it contained a part of a cDNA encoding a protein-O-mannosyl transferase and a part of the egl4 cDNA (Saloheimo et al. 1997). The mannosyl transferase-encoding part of the insert (300 bp) was digested out from the cDNA plasmid and used as a probe in the screening of a cDNA library
Table 1 Saccharomyces cerevisiae strains used for the experiments. SEY6210 and the pmt1, pmt4, pmt1,2, pmt1,4 disruptants were a gift from Dr. Sabine Strahl (Lehrstuhl fur Zellbiologie und Pflanzenphysiologie, Universitat Regensburg, Germany). BY4741 and the pmt2 disruptant were from EUROSCARF Strain
Genotype
SEY6210 pmt1D pmt4D pmt1D/pmt2D pmt1D/pmt4D BY4741 pmt2D
(Mata, ura3-52, leu2-3,112, his3D200, trp1-D901, lys2-801, suc2-D9) SEY6210(Mata, ura3-52, leu2-3,112, his3D200, trp1-D901, lys2-801,suc2-D9, pmt1::HIS3) SEY6210(Mata, ura3-52, leu2-3,112, his3D200, trp1-D901, lys2-801, suc2-D9, pmt4::TRP1) SEY6210(Mata, ura3-52, leu2-3,112, his3D200, trp1-D901, lys2-801, suc2-D9, pmt1::HIS3, pmt2::LEU2) SEY6210(Mata, ura3-52,leu2-3,112, his3D200, trp1-D901, lys2-801, suc2-D9, pmt1::HIS3, pmt4::URA3) (Mata,his 3D1, leu2D0, met15D0, ura3D0) BY4741(Mata,his 3D1, leu2D0, met15D0, ura3D0, pmt2::kanMX4)
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in lambdaZAP (Sta˚lbrand et al. 1995). Of the 500,000 bacteriophage plaques analysed, three gave a radioactive signal. The clones were excised into plasmids and one of these plasmids (pJSK2-4) was found to contain a full-length pmt1 cDNA of about 3,100 bp; and this cDNA was sequenced from both strands. The cDNA of 3,084 bp had an open reading frame of 2,319 bp, predicted to encode a protein of 773 amino acids. The translation start site was preceded by a CTrich region (109 CT in 133 nucleotides) characteristic for many fungal genes (Gurr et al. 1987). Six stop codons in the same reading frame as the start codon of the PMTI protein were found in the 5’ flanking region, showing that the isolated cDNA encoded the whole pmt1 proteincoding region. A search performed using the BLAST program (Altschul et al. 1997) through the SwissProt protein database with the amino acid sequence encoded by the open reading frame, showed 51% identity to S. cerevisiae Pmt4 protein. The predicted protein was also 53% and 33% identical to Sch. pombe Pmt1 and human POMT1, respectively. The deduced amino acid sequence of T. reesei PMTI and its alignment with C. albicans Pmt1, Sch. pombe Pmt1, S. cerevisiae Pmt1 and Pmt4 and human POMT1 are shown in Fig. 1. Using the criteria described in Girrbach et al. (2000), Trichoderma PMTI could be classified to the PMT4 family of protein O-mannosyl transferases. Protein O-mannosyl transferases are integral ER membrane proteins and, consistent with this, an analysis of T. reesei PMTI by the PSORT II (http://psort.nibb.ac.jp/) program revealed the ER retention signal XXRR (ARSP) at the N-terminus of the predicted protein. The protein was also analysed by the Tmpred program (Hoffmann and Stoffel 1993) to find potential membrane-spanning domains. Hydropathy analysis predicted PMTI to be an integral membrane protein with ten transmembrane segments concentrated at both termini and a central hydrophilic part (Fig. 2). The hydropathy profile of the Trichoderma protein is almost identical to that of S. cerevisiae Pmt1, 2, 3 and 4 proteins (Immervoll et al. 1995). The N-terminus of T. reesei PMTI is predicted to face the cytoplasm, as was shown for S. cerevisiae Pmt1. The central hydrophilic part of yeast Pmt1p has been shown to reside in the ER lumen (Strahl-Bolsinger and Scheinost 1999). The active site or substrate-binding regions of the PMT proteins are not known. The predicted Trichoderma PMTI protein contains four potential N-glycosylation sites. Three of them are located in the central hydrophilic part of the enzyme and the last one close to the C-terminus. Yeast Pmt proteins contain from two to four potential N-glycosylation sites. c
Fig. 1 The deduced amino acid sequence of the Trichoderma PMTI protein and its alignment with the sequences of Candida albicans Pmt1p (candidapmt), Saccharomyces cerevisiae Pmt1p and Pmt4p (cerpmt1, cerpmt4) Schizosaccharomyces pombe Pmt1p (pombepmt1) and human POMT1p (humanpmt1)
The sites are not conserved between yeast Pmt proteins and Trichoderma PMTI. It is known from biochemical studies that, in yeast Pmt1p, all three N-glycosylation
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After transformation with the Trichoderma pmt1 gene, the enzyme activity remained unchanged in the membrane fraction from the pmt4 and pmt1 mutants. Only slightly (by up to 20%) higher activity was observed in the double pmt1,2 mutant. The highest activation, by 73%, was observed in the pmt2 mutant transformed with the T. reesei pmt1 gene. This suggests that the Trichoderma PMTI protein is able to make an active complex with S. cerevisiae Pmt1p. In the double pmt1,2 mutant, the PMTI protein remains inactive because of the lack of a partner to make the Pmt1/PMTI complex. Protein O-mannosylation in vivo
Fig. 2 Hydropathy analysis of the predicted T. reesei protein Omannosyltransferase. The sequence was analysed using the Tmpred program (Hoffmann and Stoffel 1993). Arrowheads indicate potential N-glycosylation sites at amino acids 392, 460, 596 and 649
sites are used in vivo (Strahl-Bolsinger and Scheinost 1999). Functional analysis of T. reesei pmt1 cDNA Protein O-mannosyltransferases catalyse the transfer of a mannosyl residue from DPM to the –OH group of serine or threonine of a protein to form an O-glycosidic linkage. In S. cerevisiae, there are seven PMT genes and their gene products are very substrate-specific. To show which of the yeast genes, if any, could be replaced by the Trichoderma pmt1 cDNA, we expressed this cDNA from a multicopy plasmid with the PMA1 promoter in different S. cerevisiae pmt mutants. Protein O-mannosyltransferase activity was measured using radioactive DPM as a substrate and total membrane fraction proteins as the mannose acceptor. The cells were grown to the logarithmic phase and harvested and their membrane fractions were isolated and subjected with the PMT activity measurements. The activity from the pmt1 transformants was compared with the strains transformed with an empty plasmid (Table 2).
To demonstrate O-mannosylation in vivo, two O-glycosylated proteins, Gas1p and chitinase, were examined by immunostaining SDS-PAGE-separated proteins from membrane fractions and cell wall preparations, respectively. The plasma membrane protein Gas1p is O-mannosylated by Pmt4p and the cell wall protein chitinase by the Pmt1p/Pmt2p complex (Strahl-Bolsinger et al. 1999). If the PMT1, PMT2 or PMT4 genes could be replaced by the Trichoderma pmt1 gene, O-glycosylation of Gas1p or chitinase should be repaired at least partially in the respective transformants and the effect should be visible on a Western blot. Immunostaining analysis showed no changes in the glycosylation of Gas1p or chitinase in strains Dpmt1 and Dpmt1,2 (data not shown). A clearly visible effect for the O-glycosylation of chitinase was observed for the Dpmt2 strain transformed with the Trichoderma pmt1 gene (Fig. 3). The effect of T. reesei pmt1 on chitinase glycosylation is in agreement with the results of in vitro protein O-mannosyltransferase activity determination shown in Table 2, suggesting that Trichoderma PMTI is a functional homologue of yeast Pmt2p.
Table 2 Protein-O-mannosyltransferase activity in S. cerevisiae PMT disruptants expressing the Trichoderma pmt1 cDNA and in control strains with an empty plasmid. Results are from five parallel experiments Strain
)pmt1 (pmol/mg protein in 2 h)
+pmt1 (pmol/mg protein in 2 h)
Dpmt1 Dpmt4 Dpmt1,2 Dpmt2
12.7±2.3 21.3±2.7 11.9±2.2 28.5±3.2
14.1±2.3 21.1±0.5 14.3±2.1 49.2±4.0
Fig. 3 Western detection of chitinase synthesised in the Dpmt2 mutant (lane 1), the Dpmt2 mutant transformed with the Trichoderma pmt1 gene (lane 2) and the parent strain (lane 3). At the right, molecular weight standards (st.) are given in kiloDaltons
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The Trichoderma pmt1 cDNA was also expressed in the temperature-sensitive double mutant pmt1,4 and the resulting strain was analysed for growth at 37 C. Lack of growth at 37 C confirmed that neither PMT1 nor PMT4 could be replaced by Trichoderma pmt1 (data not shown). The functional homology of Trichoderma pmt1 to S. cerevisiae PMT4 was eliminated by in vitro activity measurement, in vivo glycosylation studies of Gas1p and growth analysis of the temperature-sensitive strain DPMT1,4 at 37 C. The latter result also eliminated functional homology to Pmt1p. In vivo analysis of chitinase O-glycosylation in the Dpmt2 strain and about 70% stimulation of protein-O-mannosyltransferase activity measured in vitro suggested the functional equivalence of Trichoderma PMTI to S. cerevisiae Pmt2p. In yeast, O-mannosylation is an essential protein modification indispensable for cell morphology and cell wall integrity (Gentzsch and Tanner 1996; Strahl-Bolsinger et al. 1999). O-linked mannose chains are required for the stability and/or correct localisation of proteins and they also affect protein function (Bourdineaud et al. 1998; Sanders et al. 1999). O-mannosylation is believed to be essential for protein secretion in T. reesei (Kubicek et al. 1987). The level of O-mannosylation can potentially influence the activity of secreted enzymes (Zakrzewska et al., manuscript in preparation) or their stability (Wang et al. 1996). Cloning and sequencing of the Trichoderma pmt1 gene will allow new experiments on the O-mannosylation process in this organism. Acknowledgements We thank Prof. Widmar Tanner and Dr. Thomas Aust for chitinase and Gas1p antibodies and Dr. Sabine Strahl for the S. cerevisiae Dpmt strains. We also thank Prof. Francis Karst for the yeast expression plasmids. This work was supported by the State Committee for Scientific Research (KBN), Warsaw, Poland, project no. 6P04B00621 grant to J.S.K.
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