Overexpression, purification, and partial characterization of Saccharomyces cerevisiae processing alpha glucosidase I

Ranjani Dhanawansa1,3, Amirreza Faridmoayer1,3, George van der Merwe3, Ying X. Li3 and Christine H. Scaman2,3

3Food, Nutrition and Health, University of British Columbia, 6650 NW Marine Drive, Vancouver, BC, V6T 1Z4, Canada

Received on December 6, 2001; accepted on January 7, 2002.


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Methods and materials
 Acknowledgments
 Abbreviations
 References
 
The gene encoding yeast processing alpha glucosidase I, CWH41, was overexpressed in Saccharomyces cerevisiae AH22, resulting in a 28-fold increase in expression of the soluble form of the enzyme. The soluble enzyme results from proteolytic cleavage between residues Ala 24 and Thr 25 of the transmembrane sequence of the membrane-bound form of the enzyme. This cleavage could be partially inhibited by addition of leupeptin and pepstatin during the enzyme isolation. The enzyme was purified to a final specific activity of 8550 U/mg protein using a combination of ammonium sulfate precipitation, anion exchange, concanavalin A, and gel filtration chromatography. The soluble form of the enzyme is a monomer with a molecular weight of 98 kDa by SDS–PAGE, and 89 kDa by gel filtration. The molecular weight decreased by approximately 5 kDa after treatment with N-glycosidase F, indicating that it is a glycoprotein. Soluble glucosidase I was sensitive to diethyl pyrocarbonate and not affected by N-ethylmaleimide, suggesting that mechanistically it is more similar to the plant than the mammalian form of the enzyme.

Key words: cloning/CWH41/glucosidase/soluble/yeast


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Methods and materials
 Acknowledgments
 Abbreviations
 References
 
Processing alpha glucosidase I (E.C. 3.2.1.106) is a key enzyme involved in the modification of oligosaccharides linked via asparagine residues to proteins. It is present in all eukaryotic cells (Dairaku and Spiro, 1997Go), with the exception of trypanosomes (Parodi, 1993Go). It catalyzes the cleavage of the terminal {alpha}(1->2) glucose residue of Glc3Man9GlcNAc2, the oligosaccharide precursor of N-linked glycoproteins, acting during protein synthesis in the endoplasmic reticulum (ER). This step is required prior to other oligosaccharide processing steps. Subsequent trimming of the oligosaccharide in the ER and cis-Golgi is catalyzed by glucosidase II and various mannosidases; additions are made to the carbohydrate structure by glycosyltransferases in the medial and trans-Golgi (reviewed by Mormen et al., 1994Go). These reactions are responsible for the wide variety of complex, hybrid, and high mannose–type N-linked oligosaccharides attached to proteins. Therefore, regulation of glucosidase I activity in essence regulates oligosaccharide processing in the cell.

Only one minor pathway present in some organisms uses an endo-alpha-mannosidase able to cleave mannose directly linked to the glucose residues and may serve to deglucosylate proteins that are not processed by glucosidase I and II. The processing of the terminal three glucose molecules are critical signals for controlling the interaction of proteins with calnexin and calreticulin to mediate the correct folding and processing of the protein (Spiro, 2000Go). In addition, glucosidase I is active on triglucosyl oligosaccharide when it is linked to dolichol phosphate prior to transfer to an asparagine residue, and it may be involved in the regulation of this compound. The biochemical importance of glucosidase is emphasized by reports of serious or fatal consequences for organisms found to be lacking glucosidase I activity (Praeter et al., 2000Go; Boisson et al., 2001Go).

Despite its importance, there is a limited understanding of the mechanism and structure of glucosidase I. Though mechanistic details of a specific glucosidase can sometimes be inferred from other members of the same family of enzyme, this is not possible for glucosidase I. This enzyme has been placed in a glycosyl hydrolase family 63 with no homology to any other glycosidase (glycosidase classification reported online at http://afmb.cnrs-mrs.fr/~pedro/CAZY).

It is known that glucosidase I from yeast (Bause et al., 1986Go), mammalian (Schweden et al., 1986Go; Romaniuk and Vijay, 1997Go), and plant sources (Zeng and Elbein, 1998Go) are comparable in substrate specificity, inhibitor sensitivity, and pH optimum (6.5–6.8). The yeast and mammalian sources show approximately 55% sequence homology, suggesting that the gene has been conserved during evolution. However, important mechanistic differences have been reported between the mammalian and plant sources of the enzyme. Mammalian forms of the enzyme are sensitive to the sulfhydryl modifying reagent N-ethylmaleimide and resistant to diethyl pyrocarbonate, a histidine-modifying reagent. Glucosidase I isolated from mung bean seedlings shows the opposite characteristics (Zeng and Elbein, 1998Go). It is not known if glucosidase I from yeast is more like the plant or mammalian enzyme.

Recently, the sequence for the yeast enzyme was reported (Romero et al., 1997Go). We report here the cloning and overexpression of CHW41, the gene encoding the membrane-bound form of Saccharomyces cerevisiae glucosidase I, and the purification and partial characterization of the soluble form of the enzyme.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Methods and materials
 Acknowledgments
 Abbreviations
 References
 
Recombinant S. cerevisiae strains, expressing soluble glucosidase I, were generated by cloning CWH41 as a 2.5-kb BglII/XhoI fragment into the BglII and XhoI sites of pHVX2 to yield pRAN1 (Figure 1). Transformed yeast showed no detrimental effects on growth rate or colony morphology but did show an increased tendency to clump during fermentation.



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Fig. 1. CWH41 was cloned as a 2.5-kb BglII/XhoI fragment into the BglII and XhoI sites of pHVX2 to yield pRAN1.

 
Levels of the soluble form of the enzyme were increased significantly in yeast transformed with pRAN1 (Table I). However, CWH41 encodes the membrane-bound form of the enzyme. This provides the first direct evidence that the soluble form of the enzyme arises from a proteolytic clipping of the membrane-bound form. N-terminal sequencing indicated that the soluble form of the enzyme resulted from proteolysis between residues Ala 24 and Thr 25, near the end of the predicted transmembrane domain of Thr 11 to Ile 28. For the mammalian enzyme, the N-terminus of the protein is suggested to be located in the cytoplasm, and the C-terminus in the lumen of the ER, with a short transmembrane segment linking the two domains (Shailubhai et al., 1991Go; Kalz-Fuller et al., 1995Go). This domain structure seems to apply to the yeast enzyme as well. A similar proteolytic cleavage of membrane-bound glycosyltransferases with release of a soluble form has been reported to occur in the Golgi (Lammers and Jamieson, 1988Go; Jaskiewicz et al., 1996Go). The proteolytic release of the soluble glucosidase I may occur when the yeast cells are broken at the beginning of the isolation protocol. Phenylmethylsulfonylfluoride (PMSF) was used as protease inhibitor in the preparation of the enzyme, suggesting that the protease involved is not a serine type. Inclusion of leupeptin and pepstatin, at 1.5 and 3.0 µg/ml, respectively, during the enzyme preparation did reduce the amount of soluble enzyme obtained from a preparation approximately twofold, but did not completely prevent its release. Addition of various protease inhibitors (ethylenediamine tetra-acetic acid, leupeptin, pepstatin A, 4-[2-aminoethyl]benenesulfonyl fluoride, trans-epoxysuccinyl-L-leucyl-amido[4-guanidino]butane [E-64],1,10 phenanthroline) did not affect the subsequent stability of the enzyme during isolation and storage and therefore were not used routinely.


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Table I. Membrane-bound and soluble glucosidase I (per gram wet weight) isolated from Saccharomyces cerevisiae transformed with pHVX2 or pRAN1
 
The membrane-bound form of the enzyme was also isolated by extracting the microsomal pellet with 1% Brij 58. Approximately 10% of the total activity was associated with the membrane-bound form of the enzyme for yeast transformed with pRAN1 (Table I). In yeast transformed with the control plasmid, pHVX2, approximately 67% of the glucosidase I activity was associated with the microsomal membranes. Therefore, overexpression of glucosidase I altered the ratio of the soluble and membrane-bound enzyme, although the levels of membrane-bound activity obtained from yeast transformed with pRAN1 and pHVX2 were not very different.

Soluble glucosidase I was isolated using a combination of ammonium sulfate precipitation, anion exchange, concanavalin A, and gel filtration chromatography (Table II). Optimization of the gradient elution of glucosidase I from the Mono Q FPLC column was critical to yield the best increase in specific activity (Figure 2). Activity calculated from the crude cell homogenate and the dialyzed ammonium sulfate precipitate were unreliable, sometimes yielding less total units of activity than the 0.2 M NaCl Toyopearl DEAE elution, possibly due to the presence of a compound(s) that influenced glucosidase I and/or the coupling enzymes, glucose oxidase and peroxidase (i.e., catalase) used in the assay. Therefore, the increase in specific activity was conservatively estimated at 57-fold, based on activity obtained from the Toyopearl elution. The purity of the enzyme is estimated at ~95%, based on band intensity after Coommassie blue staining (Figure 3). Levels of the soluble enzyme expression were increased 28-fold over that of the control, based on total activity calculated after Toyopearl anion-exchange chromatography.


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Table II. Purification of soluble glucosidase I isolated from Saccharomyces cerevisiae transformed with pRAN1a
 


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Fig. 2. Elution of glucosidase I from Mono-Q FPLC column. The peak containing glucosidase I activity is indicated by the arrow. The solid line is the absorbance at 280 nm, and the dashed line is the conductivity.

 


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Fig. 3. SDS–PAGE of soluble alpha glucosidase I after gel filtration chromatography. Lane A, glucosidase I; lane B, molecular weight standards.

 
Based on the protein sequence, the membrane-bound yeast enzyme has a predicted molecular weight of 96,512 Da, whereas the soluble truncated version isolated in this work has a predicted molecular weight of 93,800 Da. The molecular weight of soluble glucosidase I by gel filtration was 89 kDa and by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was 98 kDa, which is slightly higher than the reported molecular weight of the membrane-bound form of the enzyme of 95 kDa (Bause et al., 1986Go). Therefore, the enzyme is a monomer, in contrast to the tetramer reported for mammalian forms of the enzyme (Shailubhai et al., 1991Go; Hettkamp et al., 1984Go). The enzyme has four potential N-glycosylation sites. Treatment with N-glycosidase F was found to reduce the molecular weight of the soluble protein by approximately 5 kDa on SDS–PAGE, indicating that it is a glycoprotein.

Kojibiose ({alpha}-D-Glc1,2{alpha}-D-Glc) is not a substrate for the soluble enzyme, because incubations with 0.5–25.0 mM kojibiose over extended periods of time did not yield any free glucose. Therefore, three glucose residues, as found in the synthetic trisaccharide used in this work, are required for catalysis; however, the influence of the hydrophobic aglycone on binding cannot be discounted because a hydrophobic binding site has been suggested to be present at the active site (Bause et al., 1986Go). Kojibiose was found to be an inhibitor of the enzyme with an estimated Ki value of 0.8 mM. The Ki value for kojibiose with the membrane-bound enzyme is reported to be 55 µM, determined at an unreported level of 14C-Glc3Man9GlcNAc2 (Bause et al., 1986Go). The difference in the Ki values may be due to the use of the soluble enzyme in this work compared to the membrane-bound form. Additionally, random incorporation of 14C labeling makes it difficult to quantitate the amount of substrate in assays using the isolate natural oligosaccharide. The previously reported Ki value may therefore have been obtained at less than saturating substrate concentrations. A similar difference in Ki values for deoxynorjirmycin was noted between soluble enzyme assayed with the synthetic trisaccharide and membrane-bound enzyme assayed with 14C-Glc3Man9GlcNAc2 (Neverova et al., 1994Go).

Glucosidase I from mammalian, plant, and yeast sources are reported to be similar in substrate specificity, inhibitor sensitivity, and pH optimum. However, distinct differences between the sensitivity of mammalian and plant enzymes to N-ethylmaleimide (NEM) and diethyl pyrocarbonate (DEPC), which react specifically with cysteine and histidine residues, respectively, have been noted. Glucosidase I from mung beans exhibited sensitivity to DEPC with 2.5 mM causing 95% inhibition, and 25 mM NEM only resulted in 20% inhibition of the enzyme (Zeng and Elbein, 1998Go). However, glucosidase I from pig liver (Zeng and Elbein, 1998Go) and rat liver (Romaniuk and Vijay, 1997Go) showed the opposite trends, being more sensitive to NEM than DEPC. The yeast glucosidase I was not sensitive to NEM, as no inhibition was observed at 10 mM; however, the enzyme was inhibited by DEPC, with 50% inhibition observed at 10 mM (Table III). Therefore, the yeast enzyme appears to be more similar to the mung bean enzyme. Romaniuk and Vijay (1997)Go have suggested that Glu594 to Trp602 of the human hippocampus enzyme, which corresponds to Glu613 to Trp621 of the yeast enzyme, is the sequence containing residues critical to catalysis of glucosidase I. In the mammalian sequence, a cysteine residue is present at position 601, and it may be responsible for the sensitivity of the mammalian forms of the enzyme to NEM. There is no cysteine residue in the corresponding sequence of the yeast enzyme, which may explain the resistance of the yeast enzyme to inhibition by NEM.


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Table III. Inhibition of soluble glucosidase I by N-ethylmaleimide and diethyl pyrocarbonate
 
Mechanistically, glucosidase I catalyzes hydrolysis of the trisaccharide with inversion of configuration of the anomeric bond (Palcic et al., 1999Go). It is well accepted that inversion of configuration occurs with a single displacement catalyzed by a general acid and general base, with glutamic or aspartic acid almost invariably playing these roles (Sinnot, 1990Go). Ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDAC), a carboxyl-specific reagent, was found to inhibit glucosidase I in a time-dependent and concentration-dependent manner, with 50 mM EDAC inhibiting greater than 90% of activity after 60 min incubation at 4°C, pH 6.8 (Figure 4). Pre-incubation of enzyme with deoxynorjirmycin (DNJM) prevented the inactivation by a subsequent addition of EDAC (Figure 5). However, this protective effect was lost after dialysis to remove DNJM and addition of EDAC. Therefore, it appears that DNJM, which binds at the active site with a Ki value of 50 µM (Neverova et al., 1994Go), prevents the derivatization of a critical carboxyl group by EDAC. Control experiments incubating enzyme with either DNJM or EDAC showed that the effects of DNJM were reversible, whereas those for EDAC were not, as expected. Studies to identify this critical residue and other amino acids involved in substrate binding and catalysis are continuing.



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Fig. 4. Inhibition of soluble alpha glucosidase I at 0 (diamonds), 10 (triangles), 30 (squares), and 50 (circles) mM EDAC.

 


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Fig. 5. Percentage of initial enzyme activity remaining in samples of glucosidase I. (A) Incubation with 10 mM DNJM for 1 h at 4°C; (B) sequential treatment with 10 mM DNJM for 1 h at 4°C, followed by 50 mM EDAC for 1 h at 4°C; (C) recovery of activity after dialysis of enzyme treated sequentially with DNJM and EDAC; (D) addition of 50 mM EDAC to enzyme treated as in C; (E) 50 mm EDAC for 1 h at 4°C; (F) dialysis of enzyme treated as in C. Results presented are the average of duplicate assays. Error bars represent the range for each pair of readings.

 

    Methods and materials
 Top
 Abstract
 Introduction
 Results and discussion
 Methods and materials
 Acknowledgments
 Abbreviations
 References
 
Microbial strains and media
Escherichia coli DH5-alpha (endA1, hsdR17, supE44, thi-1 1- , recA1, gyrA96, relA1, •lacU169 [{varphi}80d lac ZDM15]) competent cells were used for E. coli transformation. The S. cerevisiae strain AH22 (MATa, leu 2-3, leu 2-112, his 4-519, canI, [cir+]) was used as the host strain for yeast transformation.

E. coli were cultured in Luria broth with or without 100 µg/ml ampicillin. S. cerevisiae were grown in YPD (Difco), or yeast nitrogen base–glucose (0.17% yeast nitrogen base without amino acids + 5% ammonium sulfate + 2% glucose + 50 mg/L histidine).

Construction of recombinant vector and transformation of yeast
YEp351 (Jiang et al., 1996Go) and S. cerevisiae genomic DNA was used as a template for the amplification of CWH41 by polymerase chain reaction (PCR). Two primers, 5'-AAT-TAG-ATC-TCG-GTT-CAG-GTT-GCT-TCA-CAA-3', and 5'-AAT-TCT-CGA-GTC-TAC-ATA-GAT-TCA-GAA-GCG-3', engineered with BglII and XhoI restriction enzyme sites, respectively, and the heat-stable DNA polymerase PWO (Roche) were used to amplify the CWH41 gene. PCR reaction conditions and amplification programs were performed according to the manufacturer’s recommendations. The resulting 2500-bp fragment was digested with BglII and XhoI and cloned into the BglII/XhoI sites of pHVX2 (Volschenk et al., 1997Go) to yield pRAN1 (Figure 1).

Plasmid manipulation and yeast and bacterial transformations
Standard procedures were used to manipulate and isolate plasmid DNA (Ausubel et al., 1994Go). Yeast (Ito et al., 1983Go) and bacterial (Inoue et al., 1990Go) transformation procedures have previously been described.

Isolation of soluble and membrane-bound glucosidase I
Yeast transformants were grown by inoculating 2 ml of an overnight culture into 250 ml fresh media. Cells were harvested at the end of the log phase after 17 h incubation at 30°C, with shaking at 300 rpm. At the time of harvest, the cultures had an A600 of approximately 4.5. Typical yield was approximately 7 g wet weight of cells per L of culture medium.

All steps for glucosidase isolation were carried out at 4°C. Cultures were centrifuged at 7000 x g for 5 min, and the cells were washed with 10 mM sodium phosphate buffer, pH 6.8 (buffer A). The cell pellet was resuspended in four volumes of the same buffer with 50 µM PMSF. At each subsequent step, 50 µM PMSF was added. Cells were broken using homogenization for 10 min with sterile glass beads (ratio of cell wet weight:beads, 1:4), with alternating 1-min intervals of vortexing and cooling on ice. The homogenate was centrifuged at 16,000 x g for 20 min to remove cell debris and glass beads. The resultant supernatant was then centrifuged at 100,000 x g for 60 min. The supernatant was used for the isolation of the soluble glucosidase I and the microsomal pellet was used for the membrane-bound form of the enzyme.

From the supernatant, a 20–60% ammonium sulfate precipitate containing soluble glucosidase I was isolated by centrifugation at 15,000 x g for 30 min. This sample was dissolved in buffer A and dialyzed for a minimum of 4 h against the same buffer. The dialyzed sample was applied to Toyopearl DEAE column (2.5 x 20 cm) equilibrated with buffer A. Unbound protein was eluted with two column volumes of buffer A. The column was then eluted with 0.1 M NaCl and 0.2 M NaCl in buffer A. Glucosidase I activity was eluted with the 0.2 M NaCl fraction, dialyzed 4 h in buffer A, concentrated using Amicon YM10 filter, and applied to FPLC using a Mono-Q HR 5/5 equilibrated with buffer A. The flow rate for FPLC was 0.2 ml/min. A continuous buffer gradient ranging from 0–1 M NaCl in buffer A was used to elute protein peaks. Activity eluted at approximately 45 ml, at 0.3 M NaCl and 30 mS conductivity. The fractions with activity (3–4 ml) were exchanged into concanavalin A loading buffer (buffer A + 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2) using an Amicon Ultrafree-MC 10,000 (Millipore) and subjected to affinity chromatography using concanavalin A Sepharose (Amersham Pharmacia Biotech) in a batch mode. Approximately 2 ml of resin was tumbled with the enzyme for 8 h. Unbound proteins were removed by repeated washing with concanavalin A loading buffer. Glucosidase I was eluted from the resin with two successive washes with five column volumes of buffer A containing 0.5 M methyl-{alpha}-D-mannoside and 0.5 M NaCl. The resin was left in contact with the buffer overnight for the first elution and 4 h for the second elution. Enzyme was then concentrated to a volume of 200 µl and chromatographed on Superdex 200 HR10/30 FPLC column (Amersham Pharmacia Biotech) at a flow rate of 0.4 ml/min, using 50 mM Na phosphate buffer, pH 6.8. Activity eluted from the gel filtration column as a single peak at approximately 12.1 ml, which corresponded to a molecular weight of 89 kDa.

Membrane-bound glucosidase I was isolated from the 100,000 x g microsomal pellet by extraction for 1 h at 4°C with 200 mM Na phosphate buffer, pH 6.8, containing 1% Brij 58, followed by centrifugation at 100,000 x g for 1 h. The supernatant was applied to the Toyopearl DEAE column, equilibrated with buffer A + 0.2% Brij 58 (buffer B). The column was washed with buffer B and buffer B + 0.1 M NaCl, and activity was eluted with Buffer B containing 0.4 M NaCl.

Enzyme assays
Glucosidase I activity was assayed using the synthetic trisaccharide {alpha}-D-Glc1,2{alpha}-D-Glc1,3{alpha}-D-Glc–O(CH2)8COOCH3 as described (Neverova et al., 1994Go). Briefly, free glucose released by glucosidase I was quantified using the coupling enzymes, glucose oxidase and peroxidase, and o-dianisidine as the chromophore. One unit of activity corresponds to the amount of enzyme that produces 1 nmol of glucose per min at 37°C, pH 6.8. The Km value for the trisaccharide was estimated from analysis of a progress curve obtained using 5 mM {alpha}-D-Glc1,2{alpha}-D-Glc1,3{alpha}-D-Glc–O(CH2)8COOCH3 (Duggleby and Clarke, 1991Go). The Ki value for kojibiose was estimated from a one-point assay at 2 mM substrate and 3.6 mM kojibiose (Segel, 1975Go).

Derivatization reactions with amino acid–specific reagents
Aliquots of enzyme were pre-incubated with 0–10 mM NEM and DEPC at room temperature for 30 min. The remaining enzyme activity was determined in the usual manner.

Glucosidase I was incubated first with 10 mM DNJM for 1 h at 4°C, followed by 50 mM EDAC with another incubation at 4°C for 1 h. The enzyme was dialyzed overnight, assayed for glucosidase activity, and treated with 50 mM EDAC again. In addition, an aliquot of enzyme was treated with 50 mM EDAC only, and activity was determined following dialysis overnight.

The reagents had no effect on the coupling enzymes used in the assay of glucosidase I.

Other methods
Protein was determined using the BioRad protein assay kit, using bovine serum albumin as the standard protein. SDS–PAGE was carried out using the method of Laemmli (1970)Go or using denaturing and reducing conditions on a 8–25% Phast Gel (Amersham Pharmacia Biotech). Protein sequence was determined by the Nucleic Acid Protein Services Unit of the Biotechnology Laboratory, University of British Columbia, using standard gas phase Edman chemistry.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Methods and materials
 Acknowledgments
 Abbreviations
 References
 
We thank H.J. van Vuuren for pHVX2 and H. Bussey for YEp35. This work was supported by an NSERC research grant to C.H.S.


    Abbreviations
 Top
 Abstract
 Introduction
 Results and discussion
 Methods and materials
 Acknowledgments
 Abbreviations
 References
 
DEPC, diethyl pyrocarbonate; DNJM, deoxynorjirmycin; EDAC, ethyl-3-(3-(dimethylamino)propyl)carbodiimide; ER, endoplasmic reticulum; NEM, N-ethylmaleimide; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonylfluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.


    Footnotes
 
1 These authors contributed equally to this paper. Back

2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Methods and materials
 Acknowledgments
 Abbreviations
 References
 
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