Effect of adding and removing N-glycosylation recognition sites on the thermostability of barley {alpha}-glucosidase

S.E. Clark1, E.H. Muslin1 and C.A. Henson1,2,3

1Department of Agronomy, University of Wisconsin–Madison and 2Cereal Crops Research Unit, Agricultural Research Service, US Department of Agriculture, 1575 Linden Drive, Madison, WI 53706, USA

3 To whom correspondence should be addressed, at the Cereal Crops Research Unit. e-mail: cahenson{at}facstaff.wisc.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The thermostability of {alpha}-glucosidase is important because the conversion of starch to fermentable sugars during the industrial production of beer and fuel ethanol typically occurs at relatively high temperatures (60–75°C). Barley (Hordeum vulgare) {alpha}-glucosidase is unstable at these elevated temperatures; however, the {alpha}-glucosidase from sugar beet (Beta vulgaris) is stable at these temperatures. An alignment of the deduced amino acid sequences of barley and sugar beet {alpha}-glucosidases revealed considerable differences in the number and position of N-glycosylation recognition sites (NGRS). Other researchers have shown that additions or removals of NGRS resulted in either the stabilization or destabilization of the enzymes at elevated temperatures. NGRS present in the barley sequence and absent in the sugar beet sequence were removed via site-directed mutagenesis from the barley protein. Recognition sites absent in the barley sequence and present in the sugar beet sequence were added via mutagenesis into the barley {alpha}-glucosidase. Two mutations significantly increased thermostability, one mutation significantly decreased thermostability and five mutations had little effect on {alpha}-glucosidase thermostability.

Keywords: Hordeum vulgare/maltase/peptide bond cleavage


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Starch is the primary source of energy and carbon in barley (Hordeum vulgare) seeds. The degradation of starch to sugar is critical for the germinating embryo until it is able to fix carbon through photosynthesis. The starch hydrolytic pathway in barley seeds consists of the concerted action of {alpha}-amylase, ß-amylase, limit dextrinase and {alpha}-glucosidase. Sun and Henson (1991Go) demonstrated that the most important enzyme during the early stages of starch degradation in germinating barley seeds was {alpha}-amylase and the second most important was {alpha}-glucosidase.

The hydrolysis of starch to simple sugars is also an important step in the production of beer, distilled beverages and ethanol fuel. The thermostability of {alpha}-glucosidase is important to fermentation industries because industrial starch hydrolysis occurs at relatively high temperatures (60–75°C). At these temperatures, {alpha}-glucosidase has been largely inactivated and its contribution to the production of fermentable sugars is limited (Muslin et al., 2000Go). The thermolability of {alpha}-glucosidase results in reduced efficiency of starch degradation and can result in an economic loss because of wasted raw materials (Muslin et al., 2003Go).

Previous studies have examined the effects of N-glycosylation on the thermostabilities of various proteins. Lige et al. (2001Go) examined the effect of the systematic removal of the three N-glycosylation recognition sites (NGRS) on the thermostability and activity of recombinant peroxidase from peanut. A NGRS is a three amino acid sequence of asparagine, then any amino acid but proline and serine or threonine (NXS or NXT) and the glycan chain is attached to the asparagine residue. Lige et al. (2001Go) determined that the removal of one of the three recognition sites significantly reduced thermostability and removal of the other two recognition sites significantly decreased activity levels of the recombinant mutant enzymes. Terashima et al. (1994Go) demonstrated that the removal of the single NGRS in rice {alpha}-amylase1A significantly decreased thermostability and altered the temperature dependence of the kinetic parameters Vmax and Km. Meldgaard and Svendsen (1994Go) compared the half-lives of Bacillus (1,3–1,4)-ß-glucanases that were produced in either Escherichia coli (no glycosylation possible) or yeast (glycosylation possible). All but one of the enzymes produced by yeast were more thermostable than the same enzymes produced by E.coli. Meldgaard and Svendsen (1994Go) proved that the glucanases produced in yeast were glycosylated and that enzymatic deglycosylation reduced the enzymes’ thermostability. Han and Lei (1999Go) showed that recombinant phytase had decreased thermostability when glycan chains were removed.

We previously reported significant variations in the thermostabilities of four plant {alpha}-glucosidases, with the {alpha}-glucosidase from barley being the most thermolabile and the sugar beet (Beta vulgaris) {alpha}-glucosidase being the most thermostable (Muslin et al., 2002Go). The barley {alpha}-glucosidase lost 90% of its activity after 10 min at 55°C. In contrast, the sugar beet {alpha}-glucosidase retained >90% of its activity after 10 min at 75°C. The present study examined the effects of adding and removing NGRS on the thermostability of barley recombinant {alpha}-glucosidase. An alignment of the deduced amino acid sequences from cDNA isolated from barley and sugar beet seeds revealed considerable differences in the number and position of NGRS. NGRS present in the barley sequence and absent in the sugar beet sequence were removed via site-directed mutagenesis from the barley protein. Recognition sites absent in the barley sequence and present in the sugar beet sequence were added via mutagenesis into the barley {alpha}-glucosidase. The thermostabilities of the resulting mutant enzymes were determined and compared with that of the recombinant wild-type enzyme.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Chemicals

Chemicals were purchased from Sigma (St Louis, MO) unless stated otherwise.

Mutagenesis and sequencing

Mutagenesis was performed according to Muslin et al. (2002Go). Generation of mutated cDNA was carried out using the primers listed in Table I to add or remove NGRS in the translated protein sequence. The mutants are identified by the original amino acid, the sequence position and the new amino acid (e.g. N298D is the mutant with the asparagine at position 298 mutated to an aspartate). The mutated cDNAs were sequenced using an automatic DNA sequencer by the Interdisciplinary Center for Biotechnology Research, University of Florida (Gainesville, FL).


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Table I. Mutagenic primers used to change nucleotide codons in the recombinant {alpha}-glucosidase cDNA sequence to add or remove N-glycosylation recognition sites in the recombinant protein
 
Expression

The mutated cDNAs were subcloned into the EcoRI site of the Pichia pastoris expression vector pPIC9K (Invitrogen, Carlsbad, CA) and transformed into P.pastoris GS115 using the Pichia EasyComp kit (Invitrogen). Histidine autotrophs were induced with methanol following the instructions in the Pichia Expression kit (Invitrogen). The recombinant wild-type and mutant {alpha}-glucosidases were secreted by P.pastoris into the culture media. Crude preparations were obtained by removing the yeast cells via centrifugation (2000 g, 10 min) and the resulting supernatants were used for thermostability studies.

Enzyme assay

{alpha}-Glucosidase activities were measured by the release of glucose from maltose. Unless stated otherwise, the enzyme was incubated for 18 h at 30°C with 20 mM maltose in 50 mM sodium succinate (pH 4.5). The glucose released was quantified by determining the reduction of NAD+ with the coupled reactions of hexokinase and glucose-6-phosphate dehydrogenase (Im and Henson, 1995Go).

Thermostability testing of wild-type and mutated {alpha}-glucosidases

Crude enzyme extracts of recombinant wild-type and mutated {alpha}-glucosidases were incubated for 10 min at temperatures from 0 to 60°C at pH 5. After cooling to room temperature, the remaining enzyme activity was assayed. We have previously demonstrated that the crude wild-type and purified wild-type recombinant {alpha}-glucosidase (rAGL) preparations had essentially the same T50 values [47.8 and 47.6°C, respectively (Muslin et al., 2002Go)], so crude enzyme preparations were used to obtain the data presented here. For statistical analysis of thermostability, a two-tailed Student’s t-test was performed using each colony as a separate replication and assuming unequal variance. The mean T50 values and standard deviations were calculated using replications of 4–16 separate colonies.

Testing of pH dependence of thermostability

Three separate extracts each of recombinant wild-type and mutated {alpha}-glucosidases were dialyzed (18 h, 4°C) against 4 l of 25 mM sodium succinate, pH 4, 5 or 6. The dialyzed enzyme extracts were incubated for 10 min at temperatures from 0 to 60°C. After cooling to room temperature, the enzyme activity was assayed. We determined that 200 mM assay buffer was required to ensure that the assay was conducted at pH 4.5 regardless of the pH of the thermostability test.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
To identify targets for mutagenesis, the deduced amino acid sequences of the barley and sugar beet seed {alpha}-glucosidases were aligned (Figure 1). There were six NGRS (NXT or NXS) in the barley deduced amino acid sequence that were not in the sugar beet sequence. There were seven NGRS in the sugar beet deduced amino acid sequence that were not in the barley sequence. Three NGRS present in the sugar beet sequence were not considered candidates for mutagenesis. One of these sites was upstream from the start of the barley {alpha}-glucosidase clone and another was in a gap in the barley sequence. The third recognition site not considered for mutagenesis was at the C-terminal end in a region of very low identity (<30%) between the barley and sugar beet sequences. The 10 NGRS selected for mutagenesis were in regions that had at least 45% sequence identity with most being in regions of >50% identity. We were unable to mutate one of the 10 NGRS targeted, possibly owing to interference from secondary structure formed by the mutagenic primer. Another NGRS mutant was not expressed because we were unable to subclone it into the P.pastoris expression vector. Results presented here are for testing the effects of mutagenesis of eight NGRS (six removed NGRS and two added NGRS; Figure 1) on the thermostability of recombinant barley {alpha}-glucosidase.



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Fig. 1. Alignment of the deduced amino acid sequences of barley (H.vulgare) and sugar beet (B.vulgaris) seed {alpha}-glucosidases. The N-glycosylation recognition sites targeted for mutagenesis are highlighted and underlined in the barley sequence.

 
The effects of the removals of the NGRS at positions 193, 391, 473 and 572 on thermostability are shown in Figure 2 and Table II. A convenient quantitative measure of thermostability is T50, the temperature at which 50% of an enzyme’s activity remains. The removal of the NGRS at positions 473 and 572 significantly (P = 0.0008 and 0.031, respectively) reduced the T50 values of the mutant enzymes with the removal at position 473 having the larger effect (Figure 2 and Table II). The T473A (threonine to alanine) enzyme lost 60% of its activity at 45°C whereas the wild-type rAGL retained 85% of its activity at that temperature (Figure 2). If the NGRS at positions 473 and 572 were glycosylated in the wild-type rAGL, the presence of the glycan chains at these positions positively affected the wild-type’s thermostability. Lige et al. (2001Go) and Terashima et al. (1994Go) also showed that the removal of NGRS decreased enzyme thermostability. The thermostability profiles and T50 values of the mutant enzymes with NGRS removed at positions 193 and 391 (S193A, serine to alanine; N391D, asparagine to aspartate) were not significantly different from the wild-type rAGL (Figure 2 and Table II). If these sites were glycosylated in the wild-type enzyme, the presence of the glycan chains did not have any significant effects on the enzyme thermostability.



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Fig. 2. Effects of removing N-glycosylation recognition sites on the thermostability of recombinant barley {alpha}-glucosidase (rAGL). Thermo stability profiles are of S193A (inverted closed triangles), N298D (closed squares), T340A (open triangles), N391D (closed triangles), T473A (closed diamonds), T572A (open squares) and wild-type rAGL (closed circles) enzymes. The points represent the means of the percentage residual activities of separate P.pastoris colonies and the error bars represent standard deviations. The enzyme extracts were incubated for 10 min at various temperatures, cooled to room temperature and assayed for 18 h for residual activity. The residual activity remaining at 40°C was set equal to 100%.

 

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Table II. T50 means and standard deviations for crude wild-type and mutant recombinant {alpha}-glucosidases (rAGL) at pH 5
 
The effect on thermostability of the removal of the NGRS present at position 298 is shown in Figure 2 and Table II. The T50 of the N298D (asparagine to aspartate) rAGL was significantly (P = 0.0005) higher than that of the wild-type rAGL (Table II). If this site was glycosylated in the wild-type enzyme, then the removal of the glycan chain may have removed any steric hinderance or chain distortion that it was causing, resulting in the N298D enzyme remaining active at higher temperatures than the wild-type enzyme.

The effect on thermostability of the removal of the NGRS present at position 340 was explored by replacing the threonine (T) with an alanine (A). The T50 of the T340A rAGL was 56.1°C, which is 7.3°C higher than that of the wild-type (Table II). The thermostability profile of this mutant shows that it retains full activity at temperatures as high as 50°C and has lost only 10–15% of its activity at 53°C (Figure 2). In contrast, the wild-type enzyme has lost ~90% of its activity at 53°C. Because the sugar beet {alpha}-glucosidase has a proline (P) in position 340, we previously removed this NGRS from the barley {alpha}-glucosidase by replacing the T at position 340 with a P (Muslin et al., 2002Go). That mutation resulted in a 10°C increase in the T50 and an enzyme that was fully stable at 55°C. Because the T340A and T340P mutations produced similar increases in enzyme thermostability, we conclude that the removal of the NGRS site was the dominant stabilizing factor and insertion of the P slightly enhanced the effect of the NGRS removal.

The effects of the additions of NGRS at positions 463 and 694 on thermostability are shown in Figure 3. The replacement of the aspartate (D) with a serine (S; D463S) had no significant impact on thermostability (Figure 3 and Table II). Either the added recognition site at position 463 was not glycosylated or the site was glycosylated and the glycan chain imparted no protection against thermal denaturation. The insertion of the NGRS at position 694 had a significant (P = 5.3 x 10–13) positive effect on the mutant enzyme’s thermostability (Figure 3). The D694N (aspartate to asparagine) rAGL had a T50 of 55.5°C whereas the wild-type rAGL had a T50 of 48.8°C (Table II). The D694N enzyme lost only 20% of its activity at 53°C compared with the >90% activity loss of the wild-type enzyme at the same temperature (Figure 3).



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Fig. 3. Effects of adding N-glycosylation recognition sites on the thermo stability of recombinant barley {alpha}-glucosidase (rAGL). Thermostability profiles are of D463A (closed squares), D463S (closed triangles), D694A (open squares), D694N (open triangles) and wild-type rAGL (closed circles) enzymes. The points represent the means of the percentage residual activities of separate P.pastoris colonies and the error bars represent standard deviations. The enzyme extracts were incubated for 10 min at various temperatures, cooled to room temperature and assayed for 18 h for residual activity. The residual activity remaining at 40°C was set equal to 100%.

 
The covalent mechanisms resulting in thermal denaturation of proteins are influenced by pH (Inglis, 1983Go; Ahern and Klibanov, 1985Go; Zale and Klibanov, 1986Go; Munch and Tritsch, 1990Go; Chen et al., 1994Go, 1995Go). At pH 4, deamidation of asparagine (N) and hydrolysis of aspartate–X (D–X) peptide bonds are the two main mechanisms contributing to thermal denaturation of many enzymes, including glucoamylases (Inglis, 1983Go; Ahern and Klibanov, 1985Go; Zale and Klibanov, 1986Go; Munch and Tritsch, 1990Go; Chen et al., 1994Go, 1995Go). At pH 6, deamidation of N, thiol-catalyzed disulfide interchange and ß-elimination of cystine have been documented to contribute to the overall thermal denaturation of some proteins (Ahern and Klibanov, 1985Go; Zale and Klibanov, 1986Go). As several of our NGRS mutations resulted in either the removal or insertion of aspartate (D) or the removal or insertion of asparagine (N), we explored the effect of pH on the thermostability of these mutant enzymes. The effects of removing NGRS by replacing the N at positions 298 and 391 with D, chosen to maintain the same approximate van der Waals volume, on enzyme thermostability as a function of pH are shown in Table III. The T50 values of both of these mutants were the same at pH 5 and 6 and both had significantly reduced T50 values at pH 4. The only difference in thermostability between these mutant enzymes and the wild-type rAGL was at pH 4, where the T50 of the N298D enzyme was 2.9°C greater than that of the wild-type. This suggests that in the microenvironment surrounding position 298, deamidation of N was more destabilizing than was hydrolysis of the peptide bond at the C-terminus of D when the protein is exposed to high temperatures at pH 4.


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Table III. Effect of pH on T50 values for wild-type and mutant recombinant {alpha}-glucosidases (rAGL): the T50 means and standard deviations are presented for pH 4, 5 and 6
 
The additions of NGRS at position 463 or 694 had different effects on enzyme thermostability as a function of pH (Table III). The T50 of the D463S mutant was the same as that of the wild-type at pH 5 and 6 yet was significantly reduced at pH 4. If position 463 is glycosylated, it is unlikely that the effect of the attached glycan chain on thermostability would be pH dependent. The substitution of the negatively charged D with the hydroxyl side chain of S may have interrupted non-covalent interactions that stabilized the protein structure at pH 4 but were not as important to thermostability at pH 5 and 6. To examine the effect of removing the D, but not adding an NGRS, on enzyme thermostability, the D463A (aspartate to alanine) enzyme was generated and its thermostability tested. The T50 of the D463A mutant was not significantly different from that of the wild-type at pH 4, but was significantly different at pH 5 and 6 (Table III). The replacement of the negatively charged D at position 463 with the hydrophobic, neutral A enabled the mutant enzyme to retain its correct structure at slightly higher temperatures than the wild-type at pH 5 and 6. In contrast to insertion of an NGRS at position 463, insertion of one at position 694 resulted in increases in T50 that were highly significant at all pHs tested (Table III). Again, the D694A enzyme was created and tested to examine the effect of removing the D without adding an NGRS on the thermostability. The T50 values of the D694A mutant were also highly significantly increased compared with those of the wild-type rAGL, suggesting that the hydrolysis of the D–X bond after position 694, and not the addition of the NGRS, was the dominant denaturing mechanism in this region at all pHs tested. The difference between the T50 values of the 694 mutants and the wild-type rAGL ({Delta}T50) was ~13–14°C at pH 4 and decreased to ~7.5°C at pH 5 and ~6°C at pH 6 (Table III). These {Delta}T50 values may indicate that a different denaturation mechanism was becoming more influential in determining the enzyme’s overall stability than the cleavage of the peptide bond after the D694 residue.

In conclusion, the effects of adding and removing NGRS were dependent on their position in the protein’s primary structure. Olsen and Thomsen (1991Go) showed that the type and position of glycosylation on the (1,3–1,4)-ß-glucanase had more of an effect on thermostability than the amount of glycosylation the protein had attached. The same may be said about the barley recombinant wild-type {alpha}-glucosidase as in several cases the removal of the potential glycosylation site increased the thermostability of the enzyme and not all potential NGRS sites may be glycosylated. In one case, the removal of the D and not the addition of the NGRS resulted in a very large increase in the thermostability of the enzyme. One possible application of this information is in enhancing yield of fermentable sugars during the malting and brewing processes, as we previously showed that the addition of a thermostable rAGL to the industrial starch hydrolysis system increased the yield of fermentable sugars (Muslin et al., 2003Go). Although transgenic barley containing a thermostable {alpha}-glucosidase gene could be generated, the use of genetically modified barley to produce malt beverages is currently not accepted. The fact that a change in only one nucleotide at either position 340 or 694 resulted in significantly increased thermostability combined with the fact that an N at 694 is found in at least two other plant species suggests that it may be worthwhile to screen H.vulgare germplasm for an {alpha}-glucosidase with either a P or A at position 340 and either an N or A at position 694.


    Acknowledgements
 
This work was supported by the US Department of Agriculture – Agricultural Research Service, the University of Wisconsin–Madison and the American Malting Barley Association, Inc. Mention of a proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other suitable products.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Muslin,E.H., Karpelenia,C.B. and Henson,C.A. (2003) J. Am. Soc. Brew. Chem., 61, 142–145.[ISI]

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Received December 16, 2003; revised February 25, 2004; accepted March 1, 2004 Edited by Valerie Daggett





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