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.
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Abstract |
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Key words: cloning/CWH41/glucosidase/soluble/yeast
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Introduction |
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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, 2000). 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., 2000
; Boisson et al., 2001
).
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., 1986), mammalian (Schweden et al., 1986
; Romaniuk and Vijay, 1997
), and plant sources (Zeng and Elbein, 1998
) are comparable in substrate specificity, inhibitor sensitivity, and pH optimum (6.56.8). 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, 1998
). 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., 1997). 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.
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Results and discussion |
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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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Kojibiose (-D-Glc1,2
-D-Glc) is not a substrate for the soluble enzyme, because incubations with 0.525.0 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., 1986
). 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., 1986
). 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., 1994
).
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, 1998). However, glucosidase I from pig liver (Zeng and Elbein, 1998
) and rat liver (Romaniuk and Vijay, 1997
) 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)
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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Methods and materials |
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E. coli were cultured in Luria broth with or without 100 µg/ml ampicillin. S. cerevisiae were grown in YPD (Difco), or yeast nitrogen baseglucose (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., 1996) 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 manufacturers recommendations. The resulting 2500-bp fragment was digested with BglII and XhoI and cloned into the BglII/XhoI sites of pHVX2 (Volschenk et al., 1997
) 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., 1994). Yeast (Ito et al., 1983
) and bacterial (Inoue et al., 1990
) 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 2060% 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 01 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 (34 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--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 -D-Glc1,2
-D-Glc1,3
-D-GlcO(CH2)8COOCH3 as described (Neverova et al., 1994
). 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
-D-Glc1,2
-D-Glc1,3
-D-GlcO(CH2)8COOCH3 (Duggleby and Clarke, 1991
). The Ki value for kojibiose was estimated from a one-point assay at 2 mM substrate and 3.6 mM kojibiose (Segel, 1975
).
Derivatization reactions with amino acidspecific reagents
Aliquots of enzyme were pre-incubated with 010 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. SDSPAGE was carried out using the method of Laemmli (1970) or using denaturing and reducing conditions on a 825% 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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