2 Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium; and 3 Department of Molecular Biomedical Research, Ghent University, Flanders Interuniversity Institute for Biotechnology (VIB), Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
Received on November 25, 2003; revised on March 15, 2004; accepted on March 15, 2004
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Abstract |
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Key words: Cel7A / endoglycosidase / N- and O-glycosylation / postsecretorial modifications / Trichoderma reesei
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Introduction |
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The glycosylation of cellobiohydrolase I (CBH I, Cel7A), a cellulase abundantly expressed by most Trichoderma reesei strains, has been studied particularly well (Table I). Trichoderma cellulases appear in several isoforms with similar catalytic and adsorption properties (Medve et al., 1998), and it has been shown that both N- and O-glycans account for the many isoforms of Cel7A (Pakula et al., 2000
).
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O-glycosylation on Cel7A was studied for T. reesei strains QM9414 and ALKO2877. Almost all threonine and serine residues in the 810-kDa linker peptide are substituted with one to three mannose residues, and the small carbohydrate-binding module present at the C- terminus is apparently not glycosylated (Harrison et al., 1998). Further structural diversification by covalent attachment of a sulfate group occurs (Harrison et al., 1998
). With the Rut-C30 strain on the other hand, the presence of a phosphorylated disaccharide was reported for Cel7A (Hui et al., 2001
), and sulfatation was observed in the linker region of acetylxylan esterase (Harrison et al., 1998
).
Thus heterogeneity due to the repartition of different glycan structures over distinct sites obviously depends on the producing strain (Stals et al., 2004) and on growth conditions (Table I). A clear knowledge of the glycosylation of endogenous proteins is mandatory, should these strains be used for heterologous expression of glycoproteins. Moreover the widespread occurrence of highly O-glycosylated linker peptides in hydrolytic enzymes implies that they fulfill an essential function. Indeed, it has been shown that these sugars contribute essentially to the stabilization of the enzyme (Neustroev et al., 1993
; Williamson et al., 1992
) and define the conformation of the linker (Receveur et al., 2002
). On the other hand, the importance of N-linked glycosylation for secretion or stability of extracellular enzymes from filamentous fungi is not clear (Boer et al., 2000
; Chen et al., 1994
; Eriksen et al., 1998
; Neustroev et al., 1993
).
A systematic analysis of Cel7A samples obtained from several T. reesei strains and grown in different media is presently described. In Part I of this study, the high-producing Rut-C30 strain is used to investigate the effect of the growth medium, and Part II focuses on strain-specific glycosylation differences.
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Results |
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Strategy in glyco-analysis
X-ray crystallography and detailed mass spectrometry (MS) analysis have previously revealed that the Cel7A catalytic module contains 10 disulfide bonds and that the N-terminal residue is pyroglutamate (Divne et al., 1994; Klarskov et al., 1997
). After correction for these posttranslational modifications, the calculated molecular mass of Cel7A core is 45,968 Da. Because N-glycosylation is restricted to Asn residues 45, 270, and 384 of the catalytic module (Klarskov et al., 1997
) and O-glycosylation to the Ser/Thr rich linker peptide (Harrison et al., 1998
), separation of these distinct domains by papain cleavage (van Tilbeurgh et al., 1986
) may allow differentiation between both glycosylation types.
The enzymatically released N-glycans were profiled by fluorescence-assisted carbohydrate electrophoresis (FACE) DNA-sequencing analyzer (DSA-FACE) (Callewaert et al., 2001) (Figure 2). Based on previous analyses, the structures can be tentatively assigned (Maras et al., 1997
). Several other analytical methods (including fractionation of neutral and charged species followed by mass determination) can be used to support these findings, as more explicitely described in the accompanying article (Stals et al., 2004
). Intact and core Cel7A proteins were also studied in detail by electrospray ionization mass spectrometry (ESI-MS). The data for core glycoprotein (Figure 3) provides further information about the number of glycosylation sites substituted with the high-mannose structures proposed (Figure 2). Whereas single GlcNAc occupation should result in a total protein mass of 46,578 Da, substitution of the main N-glycan (GlcMan7GlcNAc2) on the three Asn sites should give a total protein mass of 51,078 Da (Figure 3). Additional heterogeneity arises from variable ratios of GlcNAc to high-mannose structures and from the presence or absence of charged groups (80-Da spaced species). The glycoforms observed for the intact protein also allow the number of mannose residues in the linker region to be estimated by differentiation with those for the core proteins. The presence of charged N-glycans (ManPGlcMan7GlcNAc2) at one, two, or three sites on core Cel7A explains for the presence of several more acidic species by isoelectric focusing on polyacrylamide gel (PAG-IEF) (Figure 4). Further MS results of the separated species support these findings (Stals et al., 2004
). Additional isoforms for intact Cel7A may be due to charged O-glycans present in the linker peptide (Figure 5).
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In contrast, almost no phosphorylated oligosaccharides can be detected in the Cel7A core protein when corn steep liquor enriched medium (B) is used (Figure 2B), but a high amount of Man5GlcNAc2 is present. Figure 3B shows the molecular weight range (47,59648,244 Da) observed for these samples, indicating single N-acetylglucosamine substitution at two glycosylation sites and the presence of uncharged structures (Man5GlcNAc2 or GlcMan78GlcNAc2) at a third site. Core protein carrying only N-acetylglucosamine at the three sites (46,582 Da) can also be detected. The single isoelectric point (IP) indicates the absence of phosphodiester bonds (Figure 4, lane 4).
Minimal medium supplemented with solid CaCO3 (C) results in a different N-glycosylation profile, showing the presence of large amounts of nonglucosylated high- mannose glycans (Man58GlcNAc2) and, again, absence of phosphorylated compounds (Figure 2C). Moreover, a broad molecular weight distribution (47,60051,076 Da) was observed, and occupancy of one, two, and/or three sites with Man5678GlcNAc2 and/or GlcNAc can readily be proposed (Figure 3C). As expected, there is no MS evidence for phosphoryl groups (80-Da spacing); this is confirmed by the observation of a single IP form (Figure 4, lane 5).
In the case of the fed-batch fermentation, phosphorylated oligosaccharides were only observed within the first 24 h of cultivation (Figure 2D135). The low molecular weight values (47,72648,703 Da) observed for all glycoprotein samples (data only shown for D5) furthermore reveal that the catalytic domain carries a single oligosaccharide chain already from the beginning of growth (Figure 3D5). The presence of phosphorylated compounds (ManPGlcMan78 GlcNAc2) in the first stage (D1) was confirmed by the presence of 80-Da embedded spacing (data not shown). Therefore core Cel7A isolated from the beginning of this fed-batch cultivation consists of two IP species, but the one with the more acidic IP disappears at a later stage (Figure 4, lanes 68).
A comprehensive overview of the N-glycosylation of Cel7A observed during this study is presented in Table II. It should be noted that inductions with lactose or cellulose (Solka Floc) give identical results (data not shown).
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In contrast, for intact Cel7A isolated from rich medium (D1) six distinct bands can be observed (Figure 5, lane 4). Because the catalytic domain of Cel7A presents only two IPs (Figure 4, lane 6), the other species originate from charged groups substituted in the linker. A reduction of the number of bands is observed at later stages of cultivation (D3 and D5) (Figure 5, lanes 56) not only due to the absence of N-phosphodiester residues in the catalytic domain (Figure 4, lanes 78) but probably also due to hydrolysis of charged O-glycan residues in the linker region.
ESI-MS (Figure 6A) reveals at least 22 species spaced by 80 Da for intact Cel7A (growth condition A). The microheterogeneity is probably even larger than could be deduced from the mass spectrum; for example, di- or triphosphorylated isoforms cannot be distinguished from "neutral" or monophosphorylated forms due to the fact that the mass of two phosphates equals this of a hexose residue. Moreover, the 80-Da spacing observed can be due to the presence of phosphodiester, phosphate, or sulfate residues. The molecular mass extends from 60,405 to 62,023 Da, with a relative proportion of glycan ranging from 15.7% to 18.8% (the protein mass equals 52,205 Da). Therefore the catalytic domain substituted with three GlcMan7GlcNAc2 structures (main glycoform: 51,078 Da, Figure 3A) is connected to a linker peptide (6237 Da, as calculated from the amino acid sequence), substituted with 1929 mannose residues (insets to Figure 6A).
When analyzing intact Cel7A isolated from rich medium (D1, D3, and D5) (Figure 6), mass spectra were obtained that gradually simplify to eight peaks, each spaced by 162 Da from its neighbor. Moreover shifts of 80 Da (phosphate or sulfate) and 162 Da (mannose) occur in time probably due to hydrolysis (see later discussion). The molecular mass of the final Cel7A sample (D5) ranges from 56,895 to 58,029 Da (8.9% to 11.1% glycan), corresponding to a protein carrying one GlcMan78GlcNAc2 and two GlcNAc structures on the catalytic domain (48,090 Da, see Figure 3 D5) and 16 to 22/23 mannose residues on the linker peptide (inset to Figure 6 D5).
Enzymatic activities present in the extracellular media alter the glycosylation of secreted cellulases
Using chromogenic substrates, the presence of several extracellular hydrolases (phosphatase, endoglycosidase, -glucosidase, chitinase, and N-acetylglucosaminidase) can be demonstrated in the different growth media; no evidence for sulfatase activity is found;
-mannosidase activity is observed only in media B and C. All are optimal at pH 57 (Figure 7). In minimal medium (A), no activity can be detected, probably due to the low pH and the high proteolytic activity under these conditions. This was also indicated by a substantial loss of cellulase activity after 3 days of growth (Figure 1) and the absence of intact proteins (sodium dodecyl sulfatepolyacrylamide gel electrophoresis [SDSPAGE]) after 5 days of growth (Figure 8, lanes 1 and 2). When grown on cellulose (A'), the fungus apparently produces intact cellulases (Figure 8, lanes 3 and 4), even under the unfavorable pH conditions of the medium (Figure 1).
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Discussion |
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Because the Rut-C30 strain was used in this comparative study, we expect to find capping glucosyl moieties at the -(1
3) branch of high-mannose N-glycans (GlcMan78GlcNAc2). This modification is reported for the proteins produced by the Rut-C30 as well as RL-P37 strains (Maras et al., 1997
; Stals et al., 2004
). However nonglucosylated Man567GlcNAc2 structures were also observed, and these are most likely generated postsecretorially by
-glucosidase and
-mannosidase action. The extent of glucosylation is dependent of the pH optima of both enzymes and the pH of the respective cultivation conditions. In rich shake flask cultivation media (pH 5), for example, all deglucosylated oligosaccharides are readily converted to Man5GlcNAc2, whereas in CaCO3-supplemented cultivations (pH 7) all intermediate products (Man567GlcNAc2) can still be observed. Because the modifications were not detectable in proteins produced in the fed-batch cultivation media, these glycosidases may be secreted on aging or released by fragmentation of the mycelium when the fungus is grown in shaking flasks.
Single N-acetylglucosamine occupation on N-glycosylation sites is reported for several cellulases produced in different T. reesei strains (QM9414, ALKO2877, and Rut-C30) (Harrison et al., 1998; Hui et al., 2001
; Klarskov et al., 1997
). Clear evidence was given for the presence of a deglycosylating activity in the culture broth of strain RL-P37: Cel12A is produced as two glycoforms early in fermentation, and the deglycosylated form accumulates later (Bower et al., 1998
). Using glycosylated Asn as a test substrate, an Endo Htype activity was observed in all extracellular media (except for that obtained under minimal growth conditions). This activity was also present when the QM6A and QM9414 strains were grown in rich media. The pH profile (optimum around 5) of this deglycosylating enzyme explains the GlcNAc:GlcMan78GlcNAc2 ratio on the three N-glycosylation sites of Cel7A core when it is produced in different growth media. Fully N-glycosylated core protein (three sites) was only isolated from minimal medium (final pH 2.5). Growth in rich medium (pH 5) or CaCO3-supplemented minimal medium (final pH 7.5) resulted predominantly in glycoforms carrying, respectively, one or two high-mannose chains. Growth of the organism at the pH optimum of this endoglycosidase could therefore result in complete deglycosylation (three GlcNAc residues). However, the main glycoform isolated from the enriched cultivation media still carries at least one oligosaccharide. Thus one glycosylation site (Asn 270) seems to be selectively more resistant to hydrolysis (Hui et al., 2001
).
A study of the de novo formation of glycoproteins shows that T. reesei Cel7A matures along the biosynthetic pathway, resulting in different IP species that are secreted in the medium. Although the enzyme leaving the ER consists of a single IP, a complex pattern of more acidic species has been observed for both intra- and extracellular glycoproteins. Glycan structures are involved in the formation of these different forms (Pakula et al., 2000). Our observations show that phosphodiester residues present on N-glycans (Maras et al., 1997
; Stals et al., 2004
) and terminal phosphate (or sulfate) substituents on O-glycans (Harrison et al., 1998
; Hui et al., 2001
) are responsible for the heterogeneous isoform population (IEF) observed for T. reesei cellulases. Therefore charged groups most probably are added by transferases in a post-ER compartment (Golgi-like vesicles).
So far, no mannophosphoryl transferase could be demonstrated in T. reesei, although this type of activity, which needs the recognition of a -(1
2)-mannobiosyl residue in the substrate, has been studied in Saccharomyces cerevisiae (Karson and Ballou, 1978
). The phosphorylated N-glycans on Cel7A from T. reesei Rut-C30 show structural analogy with core phosphomannans present on the yeast cell wall (Figures 11A and B). However, two differences appear: The
1-3 arm is not phosphorylated, and the
1-6 branch carrying the phosphodiester linkage is lacking an extra
1-2 mannose in the Cel7A glycan. The capping glucose at the
1-3 arm present in the N-glycans from the Rut-C-30 strain probably prevents a second transfer, and therefore only monophosphorylated structures are observed. Indeed, with the wild type (QM6A) and with strains originating from the QM9414 lineage, we detected N-glycans carrying two phosphodiester linkages (Stals et al., 2004
). The absence of
-(1
2)-mannobiosyl linkages in Cel7A (Figure 11B) may be due to the broader specificity of the T. reesei
-(1
2)-mannosidase. The yeast enzyme can only hydrolyze the
1-2 mannosyl linkage in the middle arm (Herscovics et al., 1999
), whereas the fungal enzyme can cleave all
-(1
2)-mannosyl residues (Maras et al., 2000
; Van Petegem et al., 2001
). Because the exact cellular localization of the T. reesei
-(1
2)-mannosidase is unknown, we speculate that this enzyme may hydrolyze the product of a putative transferase at the level of the Golgi compartment or even later in the medium.
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Evidence for a highly mannosylated and charged linker peptide was also presented. This is the case in minimal medium, whereas in enriched medium the loss of several mannose residues and a simplification of the isoform pattern toward a glycoprotein devoid of charged O-glycans are observed. The presence of an extracellular -(1
2,3,6)-mannosidase was already reported (Eneyskaya et al., 1998
; Kulminskaya et al., 1998
) and could be responsible for partial postsecretorial trimming of the linker region.
The function of these phosphate groups present in Cel7A is unclear. In a number of cellular events, oligosaccharides serve as recognition markers for sorting processes, such as secretion, insertion to cell membrane, or incorporation into organelles (e.g., Man-6-P residues as key recognition signals for lysosomal protein precursors). However, because the major glycoform secreted by the fungus carries only uncharged N-glycans (Stals et al., 2004), we do not consider the occurrence of Man-6-P as a prerequisite for secretion. Alternatively, the presence of phosphorylated N-glycans on secreted proteins could be related to a cellular stress response, reminiscent of mannophosphoryl transfer to yeast cell wall mannans (Jigami and Odani, 1999
). This yeast-type response to growth phase and environmental conditions (high osmolarity) resulting in a hydration shell of the cell wall against high salt stress is probably not applicable to glycoproteins secreted by the filamentous fungus T. reesei. However, the modifications may be a stress response to the low pH conditions of the minimal medium. A systematic study of mannosylphosphate transfer induced by a variety of stress factors will lead to a further understanding of the functional role of these modifications. Ongoing research aims to identify genes encoding for manno (phosphoryl) transferring enzymes as well as for the endoglycosidase responsible for the extensive trimming observed under certain culture conditions.
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Materials and methods |
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A 5-day, 14-L fed-batch fermentation was set up using a rich medium with corn steep liquor as nitrogen source (D). Temperature was maintained at 28°C and pH at 4 (Hui et al., 2001). Samples were harvested 1, 3, and 5 days after the induction of cellulase production (D1, D3, and D5) as provided by Iogen (Ottawa, Canada).
The pH of the cultures and the activity on the chromogenic substrate CNPLac was measured as a function of time. Medium (40 µl) was incubated with 2.5 mM substrate (160 µl) dissolved in 100 mM sodium phosphate buffer, pH 5.7. Activity was followed continuously at 405 nm for 10 min with a Benchmark microtiterplate reader (Bio-Rad, Nazareth Eke, Belgium).
Purification of Ce17A by DEAE-anion exchange chromatography
Intact and core Cel7A were purified by DEAE Sepharose FF chromatography (Tomme et al., 1988). The purification was monitored by 7.5% SDSPAGE and by activity measurements on chromogenic glycosides of cellobiose and lactose. The activity against CNPLac/CNPG2 was used as an indicator of the purity of Cel7A and its contamination with Cel7B (Claeyssens and Aerts, 1992
).
Preparation of the catalytic core domain
The catalytic domain was prepared by partial proteolytic cleavage of the intact protein with papain at a substrate:enzyme ratio of 80:1 (w/w) in 50 mM NaOAc, pH 5, for 17 h at room temperature (van Tilbeurgh et al., 1986). Purification of the catalytic core and linkercarbhydrate binding domain peptides was performed by DEAE anion exchange chromatography as already described.
Fluorescent labeling and electrophoresis of N-glycans
Derivatization of PNGase F released N-glycans with 8-amino-1,3,6-pyrenetrisulfonic acid; clean-up and subsequent DSA-FACE analysis was performed (Callewaert et al., 2001). To discriminate neutral oligosaccharides from those carrying a phosphodiester bond, the derivatized N-glycans were heated in the presence of mild acid (0.02 N HCl, 100°C, 30 min) and analyzed. Faster-migrating compounds indicate the formation of a terminal phosphate residue (hydrolysis of phosphodiester bond) as evidenced by subsequent alkaline phosphatase treatment.
Analysis of glycoproteins
ESI-MS
Mass spectra were acquired on a quadrupole time-of-flight (Q-TOF) instrument (Micromass, Manchester, U.K.) equipped with a nanospray source. The samples were desalted using an ultrafree-filter, MWCO 10 kDa (Millipore), dissolved in 50% acetonitrile (0.1% formic acid) to a final concentration of 5 pmol/µl, and measured in the positive mode (needle voltage +1250 V) using Protana (Odense) needles. Mass spectra were processed using MaxEnt software. Mass accuracy was typically within 0.010.02% from the calculated value.
IEF-PAG
For IEF-PAG experiments, a PhastSystem (Amersham Biosciences, Uppsala, Sweden) was used. A dry precast homogeneous polyacrylamide gel (3.8 cm ± 3.3 cm) was rehydrated with 120 µl Pharmalyte 2.55 (Amersham Biosciences), 20 µl Servalyt 37 (Serva Electrophoresis GmbH, Heidelberg, Germany), and 1860 µl bidistilled water for 2 h. In a prefocusing step (2000 V, 2.5 mA) the pH gradient was formed, and 1 µl samples (10 mg protein/ml) were subsequently applied at the cathode position; electrophoresis was run to a final value of 450 Vh. At the end of the run, Cel7A activity was revealed by immersing the gel in 2 mM 4-methylumbelliferyl ß-lactoside (sodium acetate buffer, pH 5). Staining with Coomassie blue R-350 was according to the manufacturer's instructions. Amyloglucosidase (IP 3.5), methyl red (dye, IP 3.75), soybean trypsin inhibitor (IP 4.55), ß-lactoglobulin A (IP 5.2), and bovine carbonic anhydrase (IP 5.85) (Amersham Biosciences) were used as marker proteins.
Analysis of extracellular enzymatic activities
Measurements using chromogenic/fluorogenic substrates
1 mM of 4-nitrophenyl phosphate, 4-nitrophenyl sulfate, 4-nitrophenyl N-acetyl -D-glucosamine, 4-nitrophenyl
-D-mannopyranoside, and 4-nitrophenyl
-D-glucopyranoside (Sigma-Aldrich, Bornem, Belgium) in 50 mM phosphate-citrate, pH 28, was incubated with extracellular medium (10x concentrated using an Ultrafree filter, molecular weight cut-off 10 kDa; Millipore). Reactions were stopped with 10% Na2CO3, and absorbance was measured at 405 nm with a Benchmark microtiter plate reader (Bio-Rad). All reactions were measured over 60 min, except PNP
GlcNAc (10 min). 4-Methylumbelliferyl ß-N,N',N''-triacetylchito-trioside (1 mM; Sigma-Aldrich) was also incubated with extracellular media, and products were analyzed by thin-layer chromatography (silica, isopropyl alcohol/nitromethane/water: 50/30/20).
Measurements on enzymatically released N-glycans
Enzymatic N-deglycosylation was performed by incubating 1 U Endo H (Sigma-Aldrich) per mg Cel7A in 10 mM sodium acetate buffer, pH 5, for 17 h at 37°C. Then, three volumes of EtOH were added and pelleted proteins redissolved in water. The released sugars (supernatant) were subsequently desalted on a Carbograph column (Alltech Associates, Inc., Lokeren Belgium) (Packer et al., 1998); after extensive washing with water N-glycans were eluted with 2 ml 25% CH3CN (0.05% trifluoracetic acid). After evaporation, the N-glycans were dissolved in 500 µl water. The water used was always of Milli-Q purity.
Uncharged and charged N-glycans (1 pmol) of Cel7A (GlcMan78GlcNAc2 and (ManP)GlcMan78GlcNAc2) were derivatized with the fluorophore 8-amine-1,3,6-naphtalene-trisulfonic acid and incubated with extracellular medium C in 50 mM sodium phosphate, pH 7, and 50 mM sodium acetate, pH 5, for 3 days. The reaction mixtures were dried and loaded in 50% glycerol on a 25% PAG (AA:Bis, 1:37.5) (Jackson, 1994). A dextran ladder and the high-mannose oligosaccharides of RNase B were similarly derivatized and loaded as a reference. Gels were run on ice with a Protean III apparatus (Bio-Rad) with ice-cold running buffer for 15 min at 120 mV and 90 min at 200 mV. Photographic images could be taken directly through the assembly glass plates after irradiation with 520 nm on a photoluminager (Boehringer Mannheim, Germany).
Measurements on Glyco-Asn
150 µM Man5GlcNAc2Asn (Sigma-Aldrich) in 100 mM acetate buffer, pH 5, was incubated with extracellular medium (10x concentrated using an Ultrafree filter, molecular weight cut-off 10 kDa). After 60 min, the reactions were diluted 10-fold in Milli-Q water and analyzed by HPAEC-PAD (Dionex, Sunnyvale, CA) with an ED40 electrochemical detector (equipped with a gold working electrode). Substrate and products were separated at 1 ml/min on a CarboPac PA-100 column (40°C) using a 060 mM sodium acetate gradient in 100 mM sodium hydroxide for 35 min. Samples (20 µl) were injected using a Gilson auto-injector. Chromatographic data were analysed using Dionex Peaknet software.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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