Glycobiology Advance Access originally published online on July 13, 2005
Glycobiology 2005 15(12):1341-1348; doi:10.1093/glycob/cwj009
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Binding residues and catalytic domain of soluble Saccharomyces cerevisiae processing alpha-glucosidase I
Food, Nutrition, and Health, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
1 To whom correspondence should be addressed; e-mail: cscaman{at}interchange.ubc.ca
Received on May 18, 2005; revised on June 20, 2005; accepted on July 3, 2005
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Abstract |
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a-Glucosidase I initiates the trimming of newly assembled N-linked glycoproteins in the lumen of the endoplasmic reticulum (ER). Site-specific chemical modification of the soluble a-glucosidase I from yeast using diethylpyrocarbonate (DEPC) and tetranitromethane (TNM) revealed that histidine and tyrosine are involved in the catalytic activity of the enzyme, as these residues could be protected from modification using the inhibitor deoxynojirimycin. Deoxynojirimycin could not prevent inactivation of enzyme treated with N-bromosuccinimide (NBS) used to modify tryptophan residues. Therefore, the binding mechanism of yeast enzyme contains different amino acid residues compared to its mammalian counterpart. Catalytically active polypeptides were isolated from endogenous proteolysis and controlled trypsin hydrolysis of the enzyme. A 37-kDa nonglycosylated polypeptide was isolated as the smallest active fragment from both digests, using affinity chromatography with inhibitor-based resins (N-methyl-N-59-carboxypentyl- and N-59-carboxypentyl-deoxynojirimycin). N-terminal sequencing confirmed that the catalytic domain of the enzyme is located at the C-terminus. The hydrolysis sites were between Arg521 and Thr522 for endogenous proteolysis and residues Lys524 and Phe525 for the trypsin-generated peptide. This 37-kDa polypeptide is 1.9 times more active than the 98-kDa protein when assayed with the synthetic trisaccharide, a-D-Glc1,2a-D-Glc1,3a-D-Glc–O(CH2)8COOCH3, and is not glycosylated. Identification of this relatively small fragment with catalytic activity will allow mechanistic studies to focus on this critical region and raises interesting questions about the relationship between the catalytic region and the remaining polypeptide.
Key words:
binding residues
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catalytic domain
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chemical modification
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-glucosidase I
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Saccharomyces cerevisiae
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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The steps involved with assembly and early processing of N-linked glycoproteins in the endoplasmic reticulum (ER) are conserved between lower and higher eukaryotes (Gemmill and Trimble, 1999
; Dairaku and Spiro, 1997). Removing the distal -1,2 glucose residue is initiated by processing -glucosidase I (EC 3.2.1.106
[EC]
) immediately after the transfer of Glc3Man9GlcNAc2 to the protein (Kilker et al., 1981). This process is followed with trimming the two remaining -1,3 glucose residues by -glucosidase II (Grinna and Robbins, 1979; Michael and Kornfeld, 1980). Glucose residues of the N-linked glycan serve as signals for the protein folding and degradation system in the ER. Therefore, -glucosidase I in conjunction with -glucosidase II play an integral function in the protein quality control system (Jakob et al., 1998; Spiro, 2000).
In Saccharomyces cerevisiae, CWH41 encodes processing -glucosidase I (Cwh41p) (Romero et al., 1997) which is a member of family 63 of the glycoside hydrolases (Coutinho and Henrissat, 1999). The enzymes in this family have been shown to act with inversion of configuration (Palcic et al., 1999). Cwh41p is a type II membrane glycoprotein with a proposed domain orientation comparable to its mammalian counterpart (Shailubhai et al., 1991; Kalz-Fuller et al., 1995; Jiang et al., 1996). Sequence comparison between enzyme orthologs (i.e., human, Caenorhabditis elegans and S. cerevisiae) shows higher identity (34–49%) at the end of the C-terminus domain where the putative catalytic domain is located (Romero et al., 1997). Indeed, proteolysis of membrane-bound mammalian and soluble yeast -glucosidase I liberated catalytic active polypeptide(s) from the luminal domain (Bause et al., 1989; Shailubhai et al., 1991; Faridmoayer and Scaman, 2004).
-Glucosidase I catalysis, like other glycosidases, is controlled by carboxylic acid residues (Koshland, 1953), as previously demonstrated by selective chemical modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Dhanawansa et al., 2002). Other residues, including Arg, Cys, and Trp, were reported to be likely participants in the binding site of mammalian -glucosidase I based on chemical modification (Pukazhenthi et al., 1993; Romaniouk and Vijay, 1997). Also, mutated -glucosidase I isolated from a patient with congenital disorder of glycosylation type IIb showed that together, Arg486Thr and Phe652Leu substitutions largely inactivated the enzyme (De Praeter et al., 2000; Volker et al., 2002). Recently, mutations of highly conserved residues, Gly725 Arg in the yeast enzyme, and Ser321 Phe in enzyme of Chinese hamster ovary cells or the analogous mutation, Ser440 Phe in the human gene, were also found to impair enzyme activity (Hitt and Wolf, 2004; Hong et al., 2004). However, further kinetic and structural studies are required to determine the role of these residues.
We have previously shown that yeast -glucosidase I is sensitive to modification of His and not Cys (Dhanawansa et al., 2002), distinguishing it from the mammalian enzyme. However, involvement of other residues such as Arg, Trp, and Tyr, in catalytic activity of yeast -glucosidase I has not been investigated. Here, we report the effect of specific chemical modification of Arg, Trp, and Tyr and provide further information about the role of His in the yeast -glucosidase I. Furthermore, the isolation and partial characterization of the active polypeptide released by endogenous proteolysis and controlled trypsin hydrolysis of yeast -glucosidase I is reported.
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Site-specific modification of
-glucosidase IChemical modification using different concentrations of site selective reagents was employed to identify amino acid residues that participate in the
-glucosidase I catalytic site. Table I summarizes the results of different reagents on inhibition of the -glucosidase I activity.
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Histidine modification with diethylpyrocarbonate
Treatment of -glucosidase I with 10 mM diethylpyrocarbonate (DEPC), 25 mM 1-deoxynojirimycin (DNM), and the combined treatment of DNM followed by DEPC, reduced -glucosidase I activity by 80–90% (Figure 1). After dialysis, 78% of activity was recovered from -glucosidase I treated with DNM alone, and 54% of enzyme activity was recovered from the -glucosidase I first protected with DNM and then reacted with DEPC. However, only 13% of activity could be recovered from the enzyme treated with DEPC alone. DEPC-modified enzyme and unmodified -glucosidase I was treated with hydroxylamine (100 mM), which has been shown to restore enzyme activity by decarbethoxylation of the N-carbethoxyimidazole derivative (Christendat and Turnbull, 1996; Godavarti et al., 1996). As hydroxylamine was inhibitory to the coupling enzymes used for the activity assay, samples were exhaustively dialyzed prior to assaying for recovery of activity. Hydroxylamine was ineffective in restoring activity in the DEPC-modified enzyme (Figure 1). The increase in activity of the DNM- and DNM–DEPC-modified -glucosidase I can be attributed to the removal of residual DNM during dialysis. The failure of hydroxylamine to restore activity of the DEPC-modified -glucosidase I likely is the result of formation of N,N'-dicarbethoxyhistidine. The formation of this double substituted adduct of His in the presence of excess of DEPC is irreversible (Miles, 1977; Roosemont, 1978).
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Although DEPC reacts preferentially with unprotonated His residues at pH values less than 7, producing the mono- and dicarbethoxyimidazole derivatives with an absorbance near 240–245 nm, it can also react with Tyr residues, resulting in a reduction of absorbance at 280 nm (Miles, 1977; Roosemont, 1978). Treatment of -glucosidase I with DEPC increased absorbance at 245 nm with no decrease at 280 nm (data not shown), indicating that treatment of -glucosidase I with DEPC under the described condition was specific for His residue. Therefore, the protection of -glucosidase I with DNM against inactivation strongly suggests that a critical His residue is involved in the mechanism of -glucosidase I.
Tyrosine modification with tetranitromethane
Specific modification of Tyr residues with tetranitromethane (TNM) proceeds at alkaline pH and produces 3-nitrotyrosine as a final adduct (Sokolovsky et al., 1966). -Glucosidase I was dialyzed in 25 mM Tris–HCl, pH 8.0, and effect of different TNM concentrations (1, 5, and 10 mM) on -glucosidase I was examined (Figure 2, inset). The highest inhibitory effect was obtained by 10 mM TNM, which reduced -glucosidase I activity by 90%. However, when -glucosidase I was reacted with 25 mM DNM first followed by 10 mM TNM, 80% of enzyme activity could be recovered after dialysis (Figure 2). Although the specific activity of -glucosidase I dropped 
5 fold from its original level due to dialysis into Tris buffer, pH 8.0, DNM was still able to bind to the enzyme active site, and protect a critical Tyr residue.
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Tryptophan modification with N-bromosuccinimide
N-Bromosuccinimide (NBS) at pH less than 7 was shown to be highly selective toward Trp residue modification (Spande and Witkop, 1967). -Glucosidase I was inhibited by NBS in a concentrated dependent manner (Figure 3, inset). Enzyme treated with 50 µM NBS lost 96% of its activity, and pretreatment with 25 mM DNM was not able to prevent enzyme inactivation (Figure 3). As well, no activity was recovered after dialysis of NBS-modified enzyme (Figure 3), suggesting that the modification produced a stable adduct.
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Arginine modification with phenyl glyoxal
Modification of Arg residues using 20 mM phenyl glyoxal (PG) in 100 mM sodium bicarbonate, pH 8.0 inactivated 95% of the enzyme activity. However, with 25 mM DNM alone, only 33% of the unmodified enzyme activity could be inhibited under these conditions (data not shown). As well, the PG adduct was rather unstable, and 92% of the starting activity was recovered after dialysis with 100 mM sodium bicarbonate, pH 8.0 (data not shown). To rule out the effect of the bicarbonate buffer on enzyme activity, enzyme was treated with 20 mM PG in 100 mM phosphate buffer, pH 6.8 for 1 h at room temperature. This resulted in only a 49% loss of enzyme activity.
Isolation of active polypeptide(s) containing the catalytic domain of -glucosidase I
We previously showed that the soluble form of -glucosidase I, purified to 95% homogeneity, was endogenously cleaved to several active polypeptides after 30 days storage at 4°C (Faridmoayer and Scaman, 2004). Extending the storage time to 60 days at 4°C (Figure 4, lane 2) did not change the pattern of hydrolyzed polypeptides in comparison to 30 days (Faridmoayer and Scaman, 2004). Isolation of two polypeptides from the hydrolyzed enzyme with the approximate molecular masses of 58 kDa and 37 kDa was achieved using the inhibitor-based resin, N-methyl-N-(5'-carboxypentyl)-1-deoxynojirimycin (methyl-CP-DNM) (Figure 4, lane 3). N-terminal sequencing of the 37-kDa polypeptide band demonstrated that endogenous cleavage occurred between residues Arg521 and Thr522, a potential trypsin cleavage site. Both peptides were eluted from the resin in low yield (1.2% of applied activity) and required an extended incubation at 4°C to be generated in an uncontrolled manner by the minor impurities in the enzyme preparation. A controlled hydrolysis using modified trypsin also produced peptides of similar molecular weights (Faridmoayer and Scaman, 2004). Therefore, the formation of polypeptides during trypsin hydrolysis, and the -glucosidase I activity, was monitored at 37°C for 150 min (Figure 5A and B). Enzymatic activity increased for the first 45 min of the hydrolysis, reaching a maximum of 1.4 times the original at 45 min. The reduction of the total activity after 45 min incubation at 37°C was likely due to denaturation of active polypeptides. As hydrolysis progressed, there was an increase in the intensity of bands at 58 and 37 kDa, and a corresponding decrease in intensity of the original band at 98 kDa.
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Reducing the temperature of the trypsin hydrolysis to 20°C and extending the time to 20 h produced a mixture of polypeptides in which the 37-kDa peptide was present at a higher concentration (Figure 6), and a 1.9 increase in activity of the polypeptide mixture was observed. The -glucosidase I activity of the hydrolyzed enzyme was completely inhibited by 25 mM DNM after 10 min incubation at 37°C. Therefore, the increase in activity appears to be associated with cleavage of the intact enzyme to the 37-kDa polypeptide.
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To isolate the catalytic active polypeptide from the mixture of hydrolyzed enzyme, CP-DNM resin was used in a batch mode. The 37-kDa polypeptide was eluted from the resin (Figure 7, lane 5) with the yield of 8.8%, along with a minor 32-kDa band. N-terminal sequencing of the 37-kDa fragment demonstrated that the cleavage site of this fragment was located between residues Lys524 and Phe525. The theoretical mass of Phe525–Phe833 is 36026 Da (http://ca.expasy.org/tools/pi_tool.html), which closely matches the mass estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) (Figure 7). The 37-kDa catalytic active polypeptide was not an N-glycosylated polypeptide, as the molecular weight remained unchanged after treatment with N-glycosidase F (Figure 8). The mass of the 98-kDa enzyme decreased 4 kDa after N-glycosidase F treatment, as has been previously reported (Faridmoayer and Scaman, 2004).
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Processing
-glucosidase I removes the outermost glucose residue from newly assembled N-glycans. Despite of the highly conserved function of this enzyme and overall similarity in domain orientation, it is not known whether the orthologs are comparable in binding and catalytic residues.
We previously reported the yeast -glucosidase I is sensitive to modification of His residues (Dhanawansa et al., 2002). In this report, we extended our investigation to establish the role of His in more detail. Our evidence strongly suggests that a His residue is located near the active site, as DNM could protect against the inactivation of -glucosidase I activity with DEPC. A critical His residue is also involved in activity of the plant enzyme which is reported to be sensitive to DEPC modification (Zeng and Elbein, 1998). In contrast, the mammalian -glucosidase I is not as sensitive to DEPC modification (Romaniouk and Vijay, 1997). His residues have not been implicated as acting directly in the catalytic mechanism of glycosidases. However, they have been reported to participate in the binding site and contribute to stabilization of the transition state of several glycosidases such as beta-galactosidase (Escherichia coli) and oligo-1,6-glucosidase (Bacillus cereus) (Sogaard et al., 1993; Huber et al., 2001; Watanabe et al., 2001). Alignment of the -glucosidase I sequences from S. cerevisiae (Swiss-Prot P53008
[GenBank]
), Arabidopsis thaliana (Swiss-Prot O64796), and the putative enzyme from Oryza sativa (GenPept accession: BAB86175
[GenBank]
) using T-Coffee, Version 2.11 (Notredame et al., 2000) yielded a single conserved His residue in the catalytic region at position 686, 712, and 734, respectively (Figure 9). Interestingly, alignment of the plant and yeast glucosidase I with their mammalian counterparts (Homo sapien Swiss-Prot Q13724
[GenBank]
, Mus musculus Swiss-Prot Q9Z2W5, and Rattus rattus Swiss-Prot O88941
[GenBank]
) and the enzyme from C. elegans (Swiss-Prot Q19426
[GenBank]
) shows an Ala residue at this location. A second conserved His residue corresponding to His660 in the yeast sequence is observed when all ortholog sequences are aligned. However, the low sensitivity of the mammalian enzymes to DEPC modification makes His660 less likely to be involved in the catalytic activity of yeast and plant enzymes. Further work is required to establish whether His686 in yeast -glucosidase I is the critical residue protected from DEPC modification by DNM.
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The specific modification of Tyr residue(s) resulted in approximately a 90% loss of the total enzyme activity. DNM protected against this modification as 80% of the enzyme activity was recovered from the enzyme protected by DNM before TNM modification. This strongly suggests that the Tyr residue is located at the binding site of the enzyme. A Tyr, with hydrogen bonding to a Glu has been found to act as the nucleophile in retaining neuraminidases. It has been suggested that the phenolic hydroxyl may provide a partial negative charge stabilization for the oxocarbonium cation intermediate of the reaction (Ghate and Air, 1998). However, as of yet, no inverting enzymes have been shown to utilize Tyr/Glu as a base. Alternatively, aromatic residues can stack on the faces of the sugar residues, affecting substrate orientation (Matsui et al., 1994). Aromatic residues appear to be important for activity of mammalian -glucosidase I. Mutation of Phe652Leu resulted in inactivation of human -glucosidase I, with an accompanying loss of binding to an affinity column (Volker et al., 2002). Interestingly, this residue corresponds to Tyr668 in S. cerevisiae, Tyr669 in A. thaliana, and Phe588 in C. elegans -glucosidase I (Figure 10). Therefore, this conserved aromatic residue in the catalytic domain may be involved in stabilizing the substrate at the active site. However, it should be noted that there are several other Tyr residues in the catalytic region of the yeast enzyme that align with Tyr or Phe residues in C. elegans, A. thaliana, and O. sativa, and one of these may be involved in the binding site. Rat mammary -glucosidase I is also highly sensitive to Tyr modification, although neither DNM nor Glc3Man9GlcNAc2 protected this enzyme against TNM inactivation (Romaniouk and Vijay, 1997), suggesting a possible structural role in this ortholog.
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Modification of Trp residue(s) with NBS also inactivated the enzyme in a concentration dependent manner, but DNM was not able to protect -glucosidase I against inactivation. This suggests that Trp may be important structurally rather than catalytically. Alternatively, DNM may not completely protect the residues of the binding site. The binding site of the enzyme accommodates, and indeed requires, at least three sugar residues as the disaccharide, kojibiose (-D-Glc-1,2-D-Glc), is not hydrolyzed by the enzyme (Dhanawansa et al., 2002) but is an inhibitor (Bause et al., 1986). Although it is possible that more than one DNM residue can be accommodated in the active site, as shown for glucoamylase (Harris et al., 1993), not all the residues involved in the catalytic activity may be protected by DNM. In contrast, the mammalian enzyme was sensitive to modification of Trp residue, and the activity was protected against chemical modification by DNM (Romaniouk and Vijay, 1997).
Modification of Arg residues with PG in sodium bicarbonate (pH 8.0) and phosphate buffer (pH 6.8) gave inconclusive results. Under the condition used, the PG adduct was unstable, and DNM could not effectively bind to the enzyme. This indicated that it was not possible to protect the active site at the optimum condition for modification of Arg. Although these results do not completely exclude the possible involvement of an Arg residue at the binding site of yeast enzyme, it seems that this residue does not play a critical mechanistic role in yeast as it does for its mammalian counterpart (Romaniouk and Vijay, 1997).
Together, the results of the chemical modification suggest that the binding motif for the yeast enzyme differs from that proposed for the mammalian enzyme of Glu594–Trp602 (Romaniouk and Vijay, 1997). In yeast, the corresponding region, Glu613-Trp621, does not contain His and Tyr residues, which indicates that binding residues are not restricted to this region. Similar to the yeast enzyme, -glucosidase I from mung bean seedling, is sensitive to His and not Cys residue (Zeng and Elbein, 1998), suggesting that the binding motif may differ between lower and higher eukaryotes. Site-directed mutagenesis and detailed kinetic evaluations will be necessary to establish the identity of the critical binding residues.
However, despite these differences, some structural features appear to have been conserved between -glucosidase I from lower and higher eukaryotes. In this work, similar to other reports for mammalian enzymes (Bause et al., 1989; Shailubhai et al., 1991), proteolysis of the yeast enzyme resulted in progressive hydrolysis to a relatively small resistant polypeptide. This polypeptide, that had significantly higher activity than the intact enzyme, was comprised of approximately one third of the C-terminal protein sequence and was released by both an endogenous protease, and trypsin hydrolysis. Cleavage sites for the endogenous protease, Arg521, and trypsin, Lys524, were three residues apart suggesting that this region of the 98-kDa soluble Cwh41p may be exposed and susceptible to proteolytic cleavage. These cleavage sites are contained within a region that has no homology with the mammalian enzyme sequences.
Of the five potential N-glycosylation sites for yeast -glucosidase I, Asn42 and Asn122 have a higher probability for glycosylation (http://www.cbs.dtu.dk/services/NetNGlyc/). Two of the less likely glycosylation sites, Asn787 and Asn805, are included in the 37-kDa active polypeptide, and we have shown here that these are not N-glycosylated. In contrast, a 39-kDa catalytic active polypeptide isolated from rat -glucosidase I is predicted to be N-glycosylated at Asn654. However, deglycosylation of rat mammary gland -glucosidase I does not drastically decrease enzymatic activity (Shailubhai et al., 1991). This suggests that the N-linked oligosaccharide is not required for the activity of the catalytic domain of the yeast or mammalian enzyme. However, it is still not clear whether N-glycosylation of -glucosidase I orthologs is necessary for the polypeptide to acquire the proper conformation during biosynthesis. Over-expression of truncated forms of the enzyme will be required to determine this.
Processing -glucosidase I in all orthologs hydrolyze substrate with net inversion of configuration (Palcic et al., 1999). Regardless of the overall similarity in mechanism and domain structure, there is discrepancy between potential binding residues of orthologs, with plant and yeast exhibiting greater similarity to each other than to mammalian forms of the enzyme. The isolation and identification of catalytic active peptides from yeast -glucosidase I suggests that it is plausible to express catalytically active truncated forms of the enzyme. Further structural and kinetic characterization of native and mutant forms of -glucosidase I will provide a better understanding about the mechanism and structure of family 63 glycosidases.
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Materials and methods |
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Materials
DEPC, PG, NBS, and the glucose assay kit (GAGO-20) were obtained from Sigma (St. Louis, MO) and TNM was obtained from Aldrich (Milwaukee, WI). The synthetic trisaccharide substrate,
-D-Glc1,2-D-Glc1,3-D-Glc–O(CH2)8- COOCH3, was a gift from Dr. M. Palcic and the methyl-CP-DNM and CP-DNM was kindly synthesized by Dr O. Srivastava. Sequencing grade-modified trypsin was obtained from Promega (Madison, WI), and N-glycosidase F was obtained from Boehringer Mannheim (Mannheim, Germany). DNM-HCl salt was purchased from Genzyme (Cambridge, MA). Chemicals used for the purification of -glucosidase I were reagent grade.
S. cerevisiae overexpressing CWH41
We previously described yeast transformation with CWH41 (Dhanawansa et al., 2002), gene that encodes -glucosidase I (Romero et al., 1997). The transformed yeast were grown in 4 L medium containing 0.17% yeast nitrogen base without amino acids, 0.5% ammonium sulphate, 2% glucose, and 50 mg/L histidine in a glass stirred-tank fermenter (Virtis Co., Gardiner, NY) as described in details elsewhere (Faridmoayer and Scaman, 2004).
Purification of soluble form of yeast processing -glucosidase I
Enzyme was extracted from 40 g of freshly harvested transformed yeast. The soluble form of -glucosidase I was purified to 95% (Dhanawansa et al., 2002) and 100% (Faridmoayer and Scaman, 2004), as determined by Coomassie stained SDS–PAGE.
Enzyme assay
-Glucosidase I activity was assayed using the synthetic trisaccharide, -D-Glc1,2-D-Glc1,3-D-Glc–O(CH2)8COOCH3, and released glucose was measured by the Sigma glucose assay kit (Neverova et al., 1994). In brief, a 4 µL aliquot of enzyme solution was mixed with 1 µL of 10 mM synthetic trisaccharide and incubated at 37°C. The reaction was quenched by adding 45 µL of 1.25 M Tris–HCl, pH 7.6. The solution was then mixed with 250 µL developing solution, prepared according to the kit instructions. After 30 min incubation at 37°C in the dark, the absorbance at 450 nm was measured. One unit of enzyme activity corresponds to the amount of enzyme that produces 1 nmol of glucose per min at 37°C, pH 6.8. The effect of all chemical modification agents and buffers on the coupling enzymes was determined. In cases where the conditions inhibited the coupling enzymes, extensive dialysis in sodium phosphate buffer, 20 mM, pH 6.8 was carried out before activity determinations.
Site-specific chemical modification of -glucosidase I
Site-specific chemical modification of -glucosidase I was performed by DEPC, TNM, NBS, and PG for His, Tyr, Trp, and Arg residue, respectively. To achieve the selective modification, optimum conditions for specific modification were adapted from other works (Kochhar et al., 1992; Romaniouk and Vijay, 1997; Kaminska et al., 2003).
Endogenous hydrolysis of 95% purified -glucosidase I
The 95% purified soluble -glucosidase I was kept at 4°C for 60 days. The pattern of hydrolysis and -glucosidase I activity was monitored after 30 and 60 days by SDS–PAGE.
Trypsin hydrolysis of highly purified -glucosidase I
Pure soluble yeast -glucosidase I in 100 mM sodium phosphate buffer (pH 6.8), was mixed with the sequencing grade-modified trypsin (approximate ratio of 10:1) and incubated at 37°C for 150 min or 20°C for 20 h. Enzyme without trypsin was incubated under the same conditions. Aliquots were taken to determine the pattern of hydrolysis and activity at various times. The -glucosidase I activity was measured for the hydrolyzed enzyme and control after trypsin activity was inhibited with phenylmethanesulfonyl fluoride (PMSF) (100 µg/mL).
Isolation of catalytic active polypeptides by CP-DNM-based resins
Catalytic active polypeptides, released by endogenous and trypsin hydrolysis of the 95% and 100% -glucosidase I, were isolated by methyl-CP-DNM and CP-DNM, respectively. Active polypeptides in 100 mM sodium phosphate buffer containing 0.5 M NaCl (binding buffer) were applied to the DNM-based resin equilibrated with the same buffer. Unbound polypeptides were removed by washing with 15 bed volumes of binding buffer, and polypeptides with -glucosidase I activity were eluted with binding buffer containing 50 mM DNM. Eluted polypeptides were dialyzed overnight against 4 L of 100 mM sodium phosphate buffer before measuring activity.
Deglycosylation with N-glycosidase F
Pure -glucosidase I and the 37-kDa active polypeptide were dialyzed in 20 mM sodium phosphate buffer, pH 7.5, containing 100 µg/mL PMSF, 0.2% SDS, and 0.5% n-octylglucoside and heated for 3 min at 95°C. After cooling, N-glycosidase F (0.4 U) was added, and the samples were incubated overnight at 30°C.
Other methods
SDS–PAGE was carried out using the method of Laemmli (1970) and with the PhastGel system (Amersham Pharmacia Biotech, Baie d’Urfe, Canada). BioRad Mini Trans-Blot system (BioRad, Hercules, CA) was used to blot protein and polypeptides onto polyvinyldifluoride (PVDF) membranes. N-terminal sequencing of blotted polypeptides was done by the Nucleic Acid Protein Services Unit, Biotechnology Laboratory, University of British Columbia, using standard gas phase Edman chemistry. Protein determinations were performed with the BioRad protein assay kit using bovine serum albumin as the standard protein.
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Acknowledgments |
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We thank Drs. O. Srivastava, N. Chan, and M. Palcic for the synthesis of the DNM-based resins and synthetic trisaccharide. This work was supported by an NSERC grant to C.H.S., Adelphian scholarship and University of British Columbia Graduate Fellowship to A.F.
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Abbreviations |
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CP-DNM, N-(5'-carboxypentyl)-1-deoxynojirimycin; DEPC, diethylpyrocarbonate; DNM, 1-deoxynojirimycin; methyl-CP-DNM, N-methyl-N-(5'-carboxypentyl)-1-deoxynojirimycin; NBS, N-bromosuccinimide; PG, phenyl glyoxal; PMSF, phenylmethanesulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TNM, tetranitromethane
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References |
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