VTT Biotechnology and Food Research, PO Box 1500, FIN-02044 VTT, Finland1
Author for correspondence: Tiina M. Pakula. Tel: +358 9 45678604. Fax: +358 9 4552103. e-mail: Tiina.Pakula{at}vtt.fi
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ABSTRACT |
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Keywords: glycosylation, protein transport, two-dimensional gel electrophoresis, metabolic labelling
Abbreviations: CBHI, cellobiohydrolase I; 2D, two-dimensional; Endo H, endoglycosidase H; ER, endoplasmic reticulum; PNGase F, peptide-N-glycosidase F
a Present address: Nokia Telecommunications, PO Box 370, 00045-Nokia Group, Finland.
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INTRODUCTION |
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The strong promoter of the cbh1 gene has been commonly used for heterologous expression in Trichoderma (for a review see Penttilä, 1998 ). The abundantly expressed CBHI is encoded by a single copy gene, the regulation of which is well characterized (Ilmén et al., 1996
, 1997
; Ilmén, 1997
). The properties of CBHI are assumed to be beneficial in the different steps of protein production, and CBHI has therefore been successfully used as a fusion partner to alleviate the rate-limiting steps in production of heterologous proteins (Nyyssönen & Keränen, 1995
; Penttilä, 1998
).
The molecular mechanisms of protein secretion in the yeast Saccharomyces cerevisiae and in mammalian cells are known in great detail and the general features of the secretory pathway seem to be well-conserved in evolution. However, knowledge of the secretory pathway in filamentous fungi is rather limited despite their high capacity to produce secreted proteins and interesting multicellular filamentous morphology. Recent progress in the dissection of the fungal pathway has been reviewed by Archer & Peberdy (1997) . With the aid of information obtained from other organisms, several genes encoding factors involved in these processes have now been cloned and characterized from filamentous fungi, e.g. those encoding a component of the signal recognition particle (Thompson et al., 1995
), foldases, such as peptide disulfide isomerase (Kajino et al., 1994
; Hjort, 1995
; Lee et al., 1996
; Malpricht et al., 1996
; Ngiam et al., 1997
; Saloheimo et al., 1999
) and BiP (van Gemeren et al., 1997
; Hijarrubia et al., 1997
), as well as components from the vesicle transport machinery, such as Sar1 (Veldhuisen et al., 1997
). The actual site of protein secretion in the hypha has been located to the hyphal tips (Wösten et al., 1991
). However, in some cases the secretion has been reported to occur from subapical compartments (Keizer-Gunnink et al., 1996
; Nykänen et al., 1997
). It is even possible that different proteins are secreted through different sites on the hypha (Nykänen et al., 1997
).
The first events in N-glycosylation of the proteins and the trimming of the glycans in the endoplasmic reticulum (ER) occur in a similar fashion in yeast and animal cells (reviewed by Goochee et al., 1991 ). The glycan structures are then modified further in the Golgi complex, in animal cells to produce the complex type glycans, and in yeast to produce the highly mannosylated large glycan structures. The O-glycans of S. cerevisiae proteins consist mainly of mannose units. The first two residues are attached to the protein in the ER, and the O-linked glycan chain is further extended in the Golgi complex (reviewed by Herscovics & Orlean, 1993
). In animal cells, the O-glycosylation occurs in the Golgi complex, and the composition of the glycans is more complex. In filamentous fungi, the glycans present on the proteins seem to differ from those in both S. cerevisiae and higher eukaryotes. In Trichoderma, the glycosylation of proteins has mainly been studied by analysing the glycan structures of CBHI. CBHI has four potential N-glycosylation sites, three of which have been reported to carry glycans in strain Rut-C30. Typically, the N-glycans are high-mannose type structures of limited size, in which the mammalian type core glycan is extended with mannose and/or glucose units (Salovuori et al., 1987
; Maras et al., 1997a
, b
). In addition, some of the N-glycans have been found to carry atypical structures like terminal mannose
-1,6-phosphodiesters (Maras et al., 1997a
, b
). Different strains of T. reesei seem to display variation in the N-glycan structures synthesized; e.g. in some strains the N-glycans are reported to be composed of only a single N-acetylglucosamine (Harrison et al., 1998
; Klarskov et al., 1997
). The O-glycans of T. reesei CBHI are mainly formed of 14 mannose units, resembling the ones found in S. cerevisiae (Fägerstam et al., 1984
; Salovuori et al., 1987
; Harrison et al., 1998
).
In this study, we have developed methodology for the metabolic labelling and subsequent analysis of the maturation and secretion of proteins in Trichoderma. The techniques enable the kinetic analysis of protein synthesis and transport at a controlled physiological state of the fungus, and will be of great importance in determining the rate-limiting steps of these processes.
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METHODS |
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Steady state of the chemostat cultivation was confirmed by analysing the fungal dry weight and the lactose, phosphate and ammonium concentrations in the culture broth. Dry weight was measured by filtering and drying mycelium samples at 105 °C to constant weight (24 h). Residual lactose and glucose in the cultivations was measured using either HPLC or an enzymic test kit (Boehringer Mannheim 176303) for lactose and the GOD-Perid method (Boehringer Mannheim 124036) for glucose. The amount of phosphate was measured as described in Basset et al. (1987) . An enzymic test kit (Boehringer Mannheim 1112732) was used for measuring ammonium concentration.
Metabolic labelling of the proteins.
The metabolic labelling experiments were carried out in 25 ml aliquots transferred gently from the bioreactor into shake flasks (temperature 30 °C, stirring 210 r.p.m.). The labelling was started immediately after the transfer. When labelling cells from chemostat cultivations, fresh medium was added into the shake flasks in order to avoid changes in the concentrations of the nutrients. Fresh medium was added at intervals of 15 s during the labelling; the amount of medium added per culture volume was calculated to be the same that would be added in the chemostat during the time period (15 s). Either 5·5 or 12·5 µCi [35S]methionine (Amersham SJ 1015, in vivo cell labelling grade, 1000 Ci mmol-1, 10 µCi µl-1) was added (mg dry wt biomass)-1 (1 Ci=3·7x1010 Bq). In some cases, [14C]mannose (Amersham CFB26, 200300 mCi mmol-1, 200 µCi ml-1) was used for the labelling of the glycan structures of the proteins [3·5 µCi (mg biomass dry wt)-1]. Samples of 1 ml were collected during a time course. The mycelium in the samples was separated from the culture medium by filtering through Millipore HVLP02500 filters, which were washed with 10 ml double-distilled water and frozen immediately in liquid nitrogen. For the analysis of the labelled proteins the cells were resuspended in 20 mM N-ethylmaleimide, 10 mM NaN3 (37 mg biomass dry wt ml-1), and disrupted by sonication (8x8 s with an MSE 150 W sonicator, 18 micron amplitude, 30 s cooling on ice between the sonication cycles).
Analysis of the labelled proteins.
Two-dimensional (2D) gel electrophoresis was carried out using the Immobiline DryStrip system (Pharmacia), and sample preparation was performed essentially according to the manufacturers instructions. The mycelium samples were prepared by resuspending the lyophilized cell lysate first into 2D lysis buffer [9 M urea; 2%, v/v, Triton X-100; 286 mM ß-mercaptoethanol; 2%, v/v, Pharmalyte 310; 560 µg biomass dry weight (25 µl buffer)-1], adding 3 vols 2D sample buffer (the lysis buffer containing 0·5%, v/v, Triton X-100), and incubating the samples at room temperature for 1 h. Insoluble material was removed by centrifugation. Equal amounts of total protein from the different time points were loaded in the gels (as determined by the Bio-Rad Protein Assay kit). The amount of protein loaded was confirmed to be in a range giving a linear response between the CBHI signal and the sample volume. Typically, 20 µg intracellular protein or 3 µg extracellular protein was loaded.
The total proteins in the mycelium lysates or culture supernatants were precipitated in 20% (w/v) TCA and 0·1% (w/v) BSA overnight on ice. The precipitate was heated at 95 °C for 10 min in 5% (w/v) TCA to release labelled tRNA (Braakman et al., 1991 ), washed with 5% (w/v) TCA, and the radioactivity in the precipitate was measured by scintillation counting.
CBHI was immunoprecipitated from cellular extracts or culture supernatants using mAbs against CBHI. The epitopes of the antibodies have been mapped to amino acids 426447 located in the C-terminal part of the core domain of the protein (-CBHI 258, 261 or 271; Aho et al., 1991
). The immunoprecipitations were carried out in 800 µl 280 mM NaCl, 28 mM Tris/HCl (pH 7·5), 5·6 mM EDTA, 1·1% (v/v) Nonidet P-40. The cell lysates or samples of the culture supernatants were first incubated in the precipitation buffer for 30 min on ice. The insoluble material was removed by centrifugation for 5 min at 14000 r.p.m. at 4 °C. Protein G Sepharose 4 Fast Flow (Pharmacia) (50 µl) was added to the supernatants, and the samples were incubated with the beads with gentle mixing at 4 °C for 1 h, after which the beads were removed by centrifugation. The mAb against CBHI (78 µg
-CBHI 258, 261 or 271; Aho et al., 1991
) and 20 µl fresh Protein G Sepharose were added to the supernatants, followed by overnight incubation at 4 °C with gentle mixing. The immunoprecipitates were then washed twice with 1 ml of the precipitation buffer containing 0·1 % (v/v) Nonidet P-40, and once with 1 ml double-distilled water, and resuspended in an appropriate buffer for enzymic treatment (as described below) or into 2D gel sample buffer.
For digestion with endoglycosidase H (Endo H expressed in E. coli; Boehringer Mannheim), the immunocomplexes bound to Protein G Sepharose beads were resuspended in 50 µl 0·125 M sodium citrate (pH 5·3) and 0·2% (w/v) SDS and heated at 95 °C for 5 min. Half of the suspension was used for the digestion with 1 mU Endo H for 18 h at 37 °C, and the other half was used as a control sample. Peptide-N-glycosidase F (PNGase F expressed in E. coli; Oxford GlycoSystems) digestion of the immunoprecipitates was carried out in 20 mM sodium phosphate (pH 7·5), 50 mM EDTA, 0·5% (w/v) SDS and 5% (v/v) ß-mercaptoethanol. Before the digestion the immunocomplexes were denatured by heating for 5 min at 95 °C, and 1% (v/v) Triton X-100 was added to the samples. The digestion with PNGase F was carried out for 18 h at 37 °C (10 U PNGase F was used per 30 µl reaction mixture).
Removal of O- and N-glycans by anhydrous trifluoromethanesulfonic acid was performed using the Glycofree Deglycosylation kit (Oxford GlycoSystems). Before the deglycosylation treatment the samples of mycelium and culture supernatant were either dialysed against 0·1% (v/v) trifluoroacetic acid and lyophilized, or alternatively extracted with phenol and precipitated with acetone. The phenol extraction was carried out by adding an equal volume of phenol into the samples, vortexing for 30 s, centrifuging at 15000 g for 5 min, and collecting the organic phases. The extraction was carried out twice, and the phenol phases from the extractions were combined. Proteins were precipitated from the organic phases by adding 4 vols cold (-20 °C) acetone, incubating the samples on ice for 10 min, and centrifuging at 15000 g for 10 min. The pellets were washed with cold acetone, kept on ice for 10 min, centrifuged for 10 min, and finally dried. The deglycosylation of the proteins was confirmed using the GlycoTrack Carbohydrate Detection kit (Oxford GlycoSystems).
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RESULTS |
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For metabolic labelling, aliquots of the fungal cultivations were gently withdrawn from the bioreactor into shake flasks. The conditions during the labelling experiment were designed to mimic the conditions in the fermenter as closely as possible. Any disturbances, such as filtering or centrifuging the fungal mycelium, which have been noticed to disturb, for example, label uptake, were omitted. The labelled amino acid precursor [35S]methionine was added to the aliquots without changing the culture medium or its composition and without a chase with unlabelled compounds in order to avoid changes in the physiological state of the fungus. The labelled newly synthesized proteins were analysed during a time course from the cell extract and the culture medium. Labelling with [14C]mannose was carried out to detect glycoproteins.
Uptake of the labelled methionine occurred very efficiently. About 95% of the label was found in the cellular extracts after the first minute of labelling in repeated experiments (Fig. 1). Because of the efficient uptake of [35S]methionine, the amount of the label used was adjusted according to the biomass present in the experiments. Incorporation of [35S]methionine into the proteins started immediately after addition of the precursor, as detected by TCA precipitation of the mycelial samples. During the first 10 min of labelling, the TCA-insoluble radioactivity increased rapidly, representing a minor portion of the total cellular label pool. Towards the end of the labelling period the incorporation continued with a declining rate. After 30 min of labelling, the TCA-insoluble pool represented 60% of the total cellular radioactivity. Therefore, free intracellular label was available and there did not seem to be limitations in incorporation of [35S]methionine into the proteins in the early stages of labelling.
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Analysis of the CBHI maturation process
The use of 2D gel electrophoresis enables the detailed analysis of the different molecular forms of CBHI. The major extracellular proteins (labelled with [35S]methionine) secreted by T. reesei Rut-C30 are shown in Fig. 3(a). The spots representing CBHI, as identified using monoclonal CBHI antibodies, are indicated by a box. By silver staining it is possible to identify at least eight different pI forms of CBHI, located in the pI range of 3·54·3, in the culture medium. At the beginning of the labelling time course there is only one major nascent pI form of CBHI detectable in the cellular extract (Fig. 3b
). The full-length molecule is already detectable within 30 s of labelling, which was the first time point analysed. The additional pI forms positioned on the acidic side of the first one appear later in the intracellular maturation process of CBHI. These pI forms can first be detected after 7 min of labelling and their relative abundance increases thereafter (Fig. 3b
). The analysis did not reveal significant amounts of partially synthesized CBHI polypeptides. Their proportion is probably too low to be detected in the cell extracts analysed directly in 2D gels, and the shorter forms are not precipitated with the CBHI antibodies, whose epitope is located in the C-terminal part of CBHI.
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DISCUSSION |
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The addition of the N-linked core glycan structures on proteins occurs while the polypeptide is being translocated through the ER membrane in higher eukaryotes and yeast, and in S. cerevisiae also the initial steps of O-glycosylation are known to take place in the ER (reviewed by Herscovics & Orlean, 1993 ). The earliest forms of CBHI detected already have a clearly higher apparent molecular mass than the partially (Endo H or PNGase F treatments) or fully deglycosylated molecules. Further, the deglycosylation treatments indicated that the more acidic pI forms of CBHI are largely due to heterogeneous glycan structures generated in post-ER intracellular compartments. The N-glycan structures of CBHI produced by the strain Rut-C30 have been reported to contain phosphate groups (Maras et al., 1997a
, b
), which may affect the pI of the protein. It is, however, noteworthy that the removal of both O- and N-glycans had a much more pronounced effect on the pI forms than treatments that merely affect the N-glycans. The O-glycans of CBHI have been reported to mainly consist of the neutral carbohydrate mannose (Salovuori et al., 1987
), but the present work indicates that these glycans may also contain acidic structures that significantly lower the pI of the protein. Sulfation of the linker glycopeptide region has been reported in CBHI produced by the T. reesei strain ALKO2788 (Harrison et al., 1998
). Even the chemically deglycosylated protein appeared in the 2D gels as a spot covering a fairly broad pI range. This indicates that CBHI may also carry some other type of post-translational modification that affects the mobility of the protein in the IEF. Alternatively, the deglycosylation treatment may have slightly affected the integrity of the polypeptide itself.
By quantifying the CBHI labelled metabolically during a time course, parameters describing the different steps in protein production were deduced. A mean synthesis time of 4 min was measured for CBHI, corresponding to a translation rate of 2·1 amino acids s-1. The newly synthesized CBHI was secreted into culture medium with a minimum time of 11 min, the secretion rate being significantly lower than the synthesis rate (about 50% under the conditions used in the study). The rates of translation and intracellular transport of proteins have been addressed most thoroughly in mammalian cell systems. There, faster translation rates have been measured, e. g. 4·5 aa s-1 for influenza virus haemagglutinin in CHO cells (Braakman et al., 1991 ) and 6 aa s-1 for apolipoprotein B-100 in Hep G2 cells (Boström et al., 1986
). On the other hand, the minimum times of secretion for several mammalian proteins have been reported to be somewhat longer than that observed here, 2030 min (Boström et al., 1986
; Fries et al., 1984
; Matlin & Simons, 1983
). However, the intracellular retention half times of different mammalian glycoproteins vary to a great extent, from about 30 min to several hours (Boström et al., 1986
; Fries et al., 1984
; Lodish et al., 1983
; Yeo et al., 1985
). In addition, the protein folding, maturation and transport processes in mammalian cells are dependent on the cell type and expression system used, as well as on external parameters such as temperature and stress conditions (Braakman et al., 1991
). The variability in secretion rates can mainly be attributed to differences in retention of the proteins in the ER and their transport to the medial Golgi level (Fries et al., 1984
; Lodish et al., 1983
; Yeo et al., 1985
).
In lower eukaryotes, the kinetics of protein transport has mainly been studied in the yeast S. cerevisiae. Secretion of invertase (Novick et al., 1981 ) and Hsp150 (Jämsä et al., 1994
) has been shown to occur with somewhat faster kinetics compared to protein transport in Trichoderma, the transport time of invertase being 5 min and the half time of secretion of Hsp150 2 min. In other systems, there are relatively scarce kinetic data on protein secretion and intracellular events in protein transport that would be comparable to the present study in terms of methodology. Metabolic labelling of proteins has been applied in the study of chymoelastase Pr1 biosynthesis in the filamentous fungus Metarhizium anisopliae (St Leger et al., 1991
): the time from uptake of labelled amino acid precursor to extracellular release of the enzyme was 6·57·2 min. This is markedly faster than the corresponding time observed for CBHI in Trichoderma (13 min). Thus it seems that the rate of protein secretion by Trichoderma is faster compared to mammalian cells but somewhat slower compared to Saccharomyces, but this does not necessarily apply to all filamentous fungi.
The kinetic analysis of Trichoderma protein synthesis and secretion described here will provide tools essential for future studies on protein production in the fungus. The efficiency of protein synthesis and transport is likely to be affected by both external conditions and the physiological state of the organism. The methodology developed in this study allows analysis of these events under different cultivation conditions. The techniques can be applied to analyse the rate-limiting events in the transport process, and to compare the kinetics of production of endogenous fungal proteins and heterologous products.
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ACKNOWLEDGEMENTS |
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Received 27 July 1999;
revised 11 October 1999;
accepted 15 October 1999.