Glutathionylation inhibits catalytic activity of Arabidopsis Î²-amylase3

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Glutathionylation inhibits catalytic activity of Arabidopsis #-amylase3 but not paralog #-amylase1 Amanda R Storm, Matthew R Kohler, Christopher E. Berndsen, and Jonathan D Monroe Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01274 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

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Biochemistry

Glutathionylation inhibits catalytic activity of Arabidopsis β-amylase3 but not paralog β-amylase1 Amanda R. Storm1,†, Matthew R. Kohler1, Christopher E. Berndsen2, Jonathan D. Monroe*,1 1

Department of Biology, James Madison University, Harrisonburg, VA 22807 and 2 Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, VA 22807

KEYWORDS: β-amylase, BAM3, BAM1, glutathionylation, post-translational modification (PTM), nitric oxide, cold stress, Arabidopsis.

ABSTRACT. β-amylase3 (BAM3) is an enzyme essential for starch degradation in plant leaves and is also transcriptionally-induced under cold stress. However, we recently reported that BAM3’s enzymatic activity decreased in cold-stressed Arabidopsis leaves, although activity of BAM1, a homologous leaf β-amylase was largely unaffected. This decrease in BAM3 activity may relate to starch accumulation reported in cold-stressed plants. The aim of this study was to explore the disparity between BAM3 transcript and activity levels under cold stress and we present evidence suggesting BAM3 is being inhibited by post-translational modification (PTM). A mechanism of enzyme inhibition was suggested by observing that BAM3 protein levels remained

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unchanged under cold stress. Cold stress induces nitric oxide (NO) signaling with one result being alteration of protein activity by nitrosylation or glutathionylation through agents such as Snitrosoglutathione (GSNO). To test whether NO-induction correlates with inhibition of BAM3 in vivo, plants were treated with sodium nitroprusside, which releases NO, and a decline in BAM3 but not BAM1 activity was again observed. Treatment of recombinant BAM3 and BAM1 with GSNO caused significant, dose-dependent inhibition of BAM3 activity while BAM1 was largely unaffected. Site-directed mutagenesis, anti-glutathione Western blots, and mass spectrometry were then used to determine that in vitro BAM3 inhibition was caused by glutathionylation at cysteine433. Further, we generated a BAM1 mutant resembling BAM3 that was sensitive to GSNO inhibition. These findings demonstrate a differential response of two BAM paralogs to the Cysmodifying reagent GSNO and provide a possible molecular basis for reduced BAM3 activity in cold-stressed plants.

Introduction Starch is an important energy- and carbon-storage compound in most plants. In leaves, starch typically accumulates in chloroplasts during the day and is broken down at night, when photosynthesis is inactive, to supply sugars for local use and for export.1 Under optimal conditions, about half of the carbon fixed during the day is exported from plastids as triose phosphate, and the other half is converted to starch.2 The ability to effectively utilize starch has cumulative effects on plant growth and biomass yield.3 In Arabidopsis thaliana, β-amylase (EC 3.2.1.2; CAZy Family 14) proteins are responsible for the majority of starch degradation, with β-amylase-1 (BAM1) primarily functioning during the day in guard cells and BAM3 primarily involved in mesophyll cells during the night.4–9 We are beginning to understand some of the regulatory mechanisms for these proteins. BAM1 is known to be redox regulated at the protein level, requiring reduction of a


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Biochemistry

disulfide bond for activity.8 Recently, it was also reported that the BAM3 enzyme is inhibited by maltotriose through feedback inhibition.10 Levels of mRNA for these two principle BAMs are also known to be altered under certain stress conditions. BAM1 mRNA was shown to be induced by heat, dehydration, and osmotic stress.9,11–13 whereas BAM3 mRNA was induced by cold stress.5,11– 15

In our previous work studying the differences between BAM1 and BAM3 we noticed another intriguing instance of potential regulation. We again found that BAM3 was upregulated by cold at the mRNA level; however, BAM3 β-amylase activity was significantly lower in cold-stressed leaves than in control leaves.13 Two possibilities for this apparent disparity are either a higher rate of BAM3 protein degradation or inhibition of BAM3 catalytic activity. Cold stress in plants is known to induce nitric oxide (NO) signaling and increased levels of signaling molecules such as S-nitrosoglutathione (GSNO),16–19 which modify proteins by attaching nitrosyl or glutathionyl groups to the thiol side chain of cysteine residues.20–24 These posttranslational modifications (PTMs) can serve to protect the cysteines from irreversible overoxidation or they can alter a protein’s function.25–28 Proteomics approaches have documented a number of proteins in photosynthetic organisms that are post-translationally modified after exposure to stresses or oxidizing conditions,17,18,29–33 although, in most cases, the functional and physiological impact of these modifications remains to be determined at the individual protein level.34 One physiological change that occurs under cold stress is the accumulation of leaf starch,13,35–37 which could be a result of the observed decrease in BAM3 activity. The importance of BAM3 in starch degradation, its intriguing behavior under cold stress, and the link between cold stress and NO signaling, led us to examine whether BAM3 was subject to regulation by PTM. The previously observed decrease in BAM3 activity in cold stressed plants


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was replicated in plants where NO release was chemically induced. We were able to show that the decrease in activity in both cases was not accompanied by a corresponding decrease in BAM3 protein levels, indicating enzymatic inhibition as a probable cause. Recombinant BAM3 was strongly and rapidly inhibited by GSNO whereas BAM1 was not functionally affected. We determined that the majority of BAM3 inhibition was due to glutathionylation of Cys433, a conserved cysteine near the active site, with some contribution from additional cysteines in or near the active site. We further found that BAM1 could be engineered to have GSNO sensitivity by insertion of cysteines at locations homologous to BAM3. The combined in vivo and in vitro results of BAM3 PTM inhibition suggest a possible in vivo cellular mechanism for the observed physiological cold stress response of starch accumulation in plants.

Experimental Procedures Plant Material and Growth Conditions Arabidopsis thaliana, ecotype Columbia (Col-0), plants were grown in Sunshine Mix #3 (Sun Gro Horticulture), supplemented with macronutrients and micronutrients as previously described.38 Plants were grown in 5” pots, five plants per pot, at 22 °C with a 12/12 hr photoperiod and 130 mol/(m-2 •s) of illumination on a growth cart (Grower’s Supply Co., Ann Arbor, MI, USA). T-DNA lines were obtained from the Arabidopsis Biological Resource Center (Columbus, OH) and included bam1 (Salk_039895), bam2 (Salk_086084), bam3 (Salk_041214), and bam5 (Salk_004259). Double mutants were generated by crossing homozygous single mutants and allowing self-pollination of confirmed double heterozygotes. All mutants were verified to be homozygous by PCR as described in Monroe et al., 2014.13 For cold stress experiments plants were transferred to a walk-in 4 °C chamber for the indicated time with lighting as described above. SNP treatments were conducted on plants grown under


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Biochemistry

normal conditions. Plants were sprayed with 2 mM SNP or water (control) and then leaves were harvested after 5 hrs. β-Amylase Activity Assays with Leaf Extracts and Recombinant Proteins Leaves from three 5-6 week-old plants per replicate extract were ground in 3 volumes of Extraction Buffer (EB) (50 mM MOPS pH 7.0, 5 mM EDTA) with sand. The soluble fraction was separated by centrifugation and added to an amylase assay solution containing 50 mM MES pH 6.0 and 10 mg/mL Lintner soluble starch (Pfansteihl Laboratories). For assays using recombinant proteins, proteins were diluted in EB containing 1 mg/mL porcine gelatin (Sigma) for stabilization before adding to the assay solution. β-amylase assays were conducted at 22 °C for 20-120 min and stopped by immersion in a boiling water bath for 3 min. Reducing sugar products were then measured by the Somogyi-Nelson assay with maltose as the standard.39 Rates of activity of recombinant BAM1 and BAM3, and leaf extracts from bam5/1 and bam5/3 plants were linear for 2 hr.13 Statistical significance of leaf extract enzymatic activity was tested using a t test where p < 0.05 was considered significant. Total protein of extracts and recombinant protein concentrations were determined using the Bio-Rad Protein Assay Kit with BSA as the standard. The effect of GSNO was determined by treating the proteins with either the indicated concentration of GSNO or water for 30 min in the dark at room temperature before using the proteins in β-amylase assays as described above. To test reversal of inhibition by reduction, GSNO or water treated protein was subsequently incubated for 20 min with 5 mM DTT or 5 mM ascorbate before using the proteins in β-amylase assays. Recombinant Protein Expression and Purification Plasmids for expressing mature BAM1 (UniProt: Q9LIR6) and BAM3 (UniProt: O23553), lacking the predicted chloroplast transit peptide, 41 and 85 amino acids respectively, in E. coli


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were a gift from H. Reinhold and are described in Monroe et al. (2014).13 Plasmids were transformed into BL21+ E. coli cells for protein expression. Cells were grown to an OD (600nm) of ~0.6 in LB media supplemented with 50 µg/mL kanamycin at 37 °C. IPTG was added to a final concentration of 0.75 mM and flasks were shaken at 20 °C overnight. Cells were lysed by sonication then nickel-nitrilotriacetic acid agarose His-Bind Resin (QIAGEN) was used to purify each protein using the manufacturer’s recommendations. Eluted proteins were dialyzed against 20 mM MOPS, pH 7.2, 100 mM NaCl, 0.2 mM TCEP, and then concentrated using an Amicon Ultra4 10K filter and stored at -80 °C until used. SDS-PAGE gels of representative purifications stained with Coomassie Blue are shown in Supplemental Figure S1. Site-directed Mutagenesis All amino acid substitutions (XnC or CnX) in BAM1 and BAM3 were generated using the QuikChange mutagenesis strategy (Agilent Technologies). Briefly, mutagenesis primers were constructed containing the desired mutation flanked on either side by ~20 nucleotides complementary to the template sequence. Primer sequences are provided in Table S1. The pET29a plasmids with wild type BAM1 and BAM3 were used as the templates. Mutagenesis primers were used to perform PCR to amplify the entire plasmid. The PCR protocol, which was adjusted according to primer melting point temperature, was as follows: 95 °C for 5 min, cycle of 95 °C for 30 sec, 60 °C for 30 sec, 72 °C for 4 min repeated 20 times, and final extension 72 °C for 10 min. PCR products were treated with DpnI for 1.5 hr at 37 °C and then transformed into DH5α E. coli competent cells. All amino acid substitutions were confirmed by sequencing of the plasmid insert at Eurofins Genomics. All mutant proteins were assayed for functionality by comparing β-amylase activity to wild-type protein. Trypsin Digest and Mass Spectrometry


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Biochemistry

Recombinant protein (0.01 mg/mL) in EB (pH 7.0) was treated with 0.5 mM GSNO or water for 30 min in the dark (inhibition is complete after 1 min), followed by treatment with iodoacetamide (IAM, 40 mM) for 30 min in the dark to block remaining cysteines by alkylation. The proteins were denatured with 0.1 % SDS at 60 °C for 1 hr. Spin columns (Pierce Concentrator, PES) were used to exchange the buffer for 50 mM Tris pH 8.0 and to concentrate the protein to ~2 mg/mL. The protein was digested with Trypsin (1:45 protease:protein, Pierce MS-grade) at 37 °C for 1215 hours. Samples were filtered and run on an Agilent 1290 Infinity HPLC-ESI-TOF mass spectrometer using a 2.1 mm Discovery BIO Wide Pore C5 column (Sigma-Aldrich). Protein samples (10 µL injected) were eluted with a gradient of 98 % Solvent A (0.1 % formic acid in water) and 2 % Solvent B (0.1 % formic acid in acetonitrile) to 10 % A and 90 % B over 10 min with a flow-rate of 0.4 mL/min. Mass spectra were acquired in positive mode from m/z 200 to 1700 at 3 specta/sec. These conditions ensure the removal of any non-covalently attached GSH and the pH of reaction was selected to selectively label cysteine thiol side chains.40 Peptide masses were calculated using the PeptideMass tool (ExPASy). Glutathionylation of a cysteine in a peptide is indicated by a 305 Da increase in mass and alkylation of free cysteines by IAM is indicated by an increase of 57 Da. The BAM3 homology model was generated by fitting the mature BAM3 amino acid sequence (lacking the 85 amino acid predicted chloroplast transit peptide) onto soybean BAM5 crystal structures (PDB ID 1Q6C and 1WDR) using I-TASSER. The structure of glutathionylated BAM3 was generated by extracting glutathione (GSH) from structure 2NTO and manually positioning it in YASARA proximal to Cys433 of the Bam3 model. The disulfide bond was built and the structure energy minimized in YASARA. Western Blot Analysis of Leaf Extracts and Glutathionylated Protein


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Recombinant protein (0.01 mg/mL) was treated with 0.5 mM GSNO or water for 30 min in the dark followed by treatment with iodoacetamide (40 mM) for 30 min in the dark then concentrated using spin columns. Protein concentration was measured using the Bio-Rad Protein Assay Kit. An equal volume of non-reducing 2x Sample Loading Buffer (8 M urea, 0.1 M Tris pH 6.8, 5 % SDS, 30 % glycerol, 0.1 % Bromophenol blue) was added and samples were boiled for 5 min. Protein samples (0.5 ug loaded) were separated by non-reducing SDS-PAGE, transferred to nitrocellulose, and blotted by overnight incubation with an anti-GSH monoclonal primary antibody (Virogen, 1:7500 titer) and 1 hour incubation with anti-mouse IgG-HRP fusion secondary antibody (LiCor, 1:6000 titer). Chemiluminescence was detected with Li-Cor WesternSure ECL substrate. A separate gel containing identical samples was stained by Coomassie Blue. Gel images were collected using a BioRad Imager. Western blots for detecting BAM3 in plant extracts were conducted using polyclonal primary antibodies obtained as a generous gift from Dr. Samuel Zeeman. Plant extracts were prepared by grinding a 0.8 cm disc of fresh leaf tissue in a 1.5 mL tube in the presence of 40 µL of EB and 40 µL 2x non-reducing Sample Loading Buffer. Tubes were boiled for 10 min and spun to pellet solids. Supernatants (5 µL) were loaded onto non-reducing gels and separated by SDS-PAGE on a 10 % Tris-Glycine gel, transferred to nitrocellulose (LiCor), and blotted with polyclonal primary antibodies against BAM3 (1:2500 titer) and anti-rabbit IgG-HRP fusion secondary antibody (Agrisera, 1:24,000 titer). Chemiluminescence was detected with Li-Cor WesternSure ECL substrate. This was repeated for leaf discs from three separate plants subjected to each treatment.

Results


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Biochemistry

In Monroe et al., 2014,13 we reported the differential effect of temperature and pH on the activity of the primary starch-degrading BAMs in Arabidopsis, BAM1 and BAM3. During this work we observed that, although BAM3 transcript levels increased under cold stress, as reported by other groups,5,11,12,14,15 the β-amylase activity attributable to BAM3 in plants lacking BAM1 and BAM5, bam5/1, actually decreased by over 50 % in plants cold stressed for four days. The contrast of increased mRNA levels and decreased enzyme activity for BAM3 suggested a hitherto unknown mechanism of BAM3 protein regulation. Observed decrease in BAM3 activity under cold-stress could be result of PTM The majority of β-amylase activity in leaf extracts is due to the catalytic β-amylases BAM5, BAM3, and BAM1,4,13 so β-amylase activity in bam5/1 plants can be mainly attributed to BAM3 and likewise β-amylase activity in bam5/3 plants can be attributed to BAM1.13 The effect of cold stress on the respective BAM3 and BAM1 activity was tested by subjecting BAM knockout plants to either 4 days at 4 °C or 4 days at room temperature as a control. Total β-amylase activity for stressed and control plants was determined by measuring formation of the product, maltose, using the Somogyi-Nelson assay

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for reducing sugars, normalizing by total protein (Figure 1A). As

previously observed,13 β-amylase activity in the cold stressed bam5/1 mutant leaf extracts had about 30 % activity of control plants. BAM1 activity levels in the bam5/3 mutant were largely unaffected under these same conditions. As numerous studies have found that BAM3 mRNA levels increase under cold stress, the decrease in enzyme activity observed under cold stress could be due to increased protein degradation or increased inhibition of enzyme activity. To test for the possibility of increased degradation of BAM3 protein, we performed Western blots to probe total BAM3 protein levels in leaf extracts from wild type and bam5/1 Arabidopsis plants subjected to either cold stress or control


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treatment. A 0.8 cm diameter leaf disc was cut from the center of a leaf and protein extracted by grinding and boiling the extract in non-reducing loading buffer. The amount of BAM3 protein detected did not decrease in cold stressed plants and protein levels may even be a little higher than in control plants, although these were not quantitative blots (Figure 1B). This indicates that BAM3 degradation under cold stress can not sufficiently explain the lower BAM3 β-amylase activity. Similar results were seen for leaf extracts from wild type and bam5/1 plants, so lack of BAM1 and BAM5 does not change the BAM3 protein expression pattern under these conditions. Another possibility for decreased β-amylase activity under cold stress is that BAM3 activity is being inhibited in some way, potentially by post-translational modification (PTM). Cold stress response in plants is known to involve nitric oxide (NO) signaling, with one outcome being the PTM of many proteins by addition of either a nitrosyl group (S-nitrosylation) or a glutathionyl group (S-glutathionylation) to the thiol group of cysteine residues. It is possible that BAM3 is a target for NO-induced PTM as part of a plant’s cold stress response. NO signaling in plants can be stimulated without cold temperatures by spraying plants with the NO-releasing compound Snitroprusside (SNP).19,41,42 To observe whether inducing NO-signaling also resulted in decreased BAM3 β-amylase activity, we sprayed bam5/1 and bam5/3 mutant plants with SNP or water and then tested leaf extracts for total β-amylase activity and BAM3 protein levels (Figure 1C, D). The total β-amylase activity in the SNP-treated bam5/1 leaves decreased to almost 60 % of the control whereas there was no difference between the treated and control bam5/3 leaves. Also, no significant changes in total BAM3 protein levels were observed in the treatment compared to the control extracts. These results led us to consider whether the BAM3 protein was being inhibited under cold stress via PTM by an NO-induced mechanism.


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Biochemistry

Figure 1. Effect of cold stress and sodium nitroprusside (SNP) treatment on β-amylase activity and BAM3 protein levels in crude leaf extracts from 5-week old Arabidopsis plants. (A) Total amylase activity in bam5/1 and bam5/3 leaves from plants incubated at normal 22 °C (grey bars) or stressed 4 °C (black bars) temperatures under a 12/12 hour photoperiod for four days. Activity assays of leaf extracts were conducted at 22 °C with soluble starch as the substrate. Each graph represents developmentally matched plants. Each replicate included all leaves from three plants. Values are means +/- SD, n=3. Statistical significance: ** p < 0.01 (compared to control), * p < 0.05 (compared to control). (B) Western blots against BAM3 in total protein extracted from 0.8 cm diameter leaf discs of Wild Type and bam5/1 plants treated as described in (A). Equal volumes of disc extract supernatant were combined with non-reducing sample loading buffer, run on a 10 % non-reducing Tris-Glycine gel, and transferred to nitrocellulose for Western blotting with antiBAM3 antibodies. Representative of results from 3 separate plants for each treatment. (C) Total


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amylase activity measured as described in (A) for plants sprayed either with water (Control, grey bars) or with 2 mM SNP (black bars) and leaves harvested 5 hours after treatment. (D) Western blot against BAM3 in total protein extracted from 0.8 cm leaf discs of bam5/1 plants treated with (+) and without SNP as described in (C). Sample preparation and analysis as described in (B).

BAM3 activity is inhibited in vitro by GSNO, likely through glutathionylation NO signaling is known to cause distinct protein PTMs.21,43–46 GSNO is an important mediator of NO signaling in cells that can cause protein nitrosylation or glutathionylation or both.24,47,48 We tested the effect of GSNO treatment on β-amylase activity of purified BAM3 and BAM1 to see if either protein was susceptible to PTM. Recombinant BAM1 and BAM3 were over-expressed and purified using His-tag affinity chromatography (Supplemental Figure S1). The proteins were pretreated with GSNO for increasing periods of time and the β-amylases’ enzyme activity was determined by measuring formation of the product, maltose, using soluble starch as the substrate as described in Experimental Procedures. Recombinant BAM3 activity was inhibited by up to 80 % when pretreated with GSNO for as little as 1 min (Figure 2A). It was found that treatment with even low levels of GSNO (0.1 mM) caused a 50 % inhibition of β-amylase activity in BAM3 relative to the untreated control but there was minimal effect on BAM1 activity (less than 20 % inhibition) with up to 2 mM GSNO (Figure 2A insert). When GSNO-treated BAM3 was incubated with DTT, which reverses both nitrosylation and glutathionylation PTMs, it resulted in restoration of about 90 % of untreated BAM3 activity, indicating that the observed inhibition by GSNO treatment was due to PTM (Figure 2B). GSNO is capable of adding either a nitrosyl or a glutathionyl group to select cysteine residues, depending on as-yet undetermined features of the microenvironment surrounding the cysteine.21,45,48,49 As


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Biochemistry

GSNO can cause both nitrosylation and glutathionylation, we next wanted to determine which modification was responsible for the observed inhibition of BAM3. Ascorbate selectively reverses nitrosylation without affecting glutathionylation50,51 so BAM3 treated with GSNO was incubated with 5 mM ascorbate before testing BAM3 activity. The ascorbate treatment did not have any effect on BAM3’s GSNO-inhibition (Figure 2B), suggesting that glutathionylation rather than nitrosylation is responsible for the inhibition of BAM3 activity. Recombinant BAM3 was also insensitive to treatment with SNP, a reagent that can directly nitrosylate but not glutathionylate proteins (data not shown). Additionally, Western blots of pure BAM3, either untreated or treated with GSNO, using anti-GSH antibodies showed that at least one glutathionyl (GSH) group was attached to BAM3 by GSNO treatment (Figure 2C).


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Figure 2. BAM3 but not BAM1 is reversibly inhibited in vitro by GSNO-induced PTM. (A) Recombinant BAM3 β-amylase activity after treatment with 0.5 mM GSNO for increasing amounts of time. Insert, activity of BAM3 (grey circles) and BAM1 (black circles) after treatment with increasing concentrations of GSNO (0.1 to 2.0 mM) for 30 min. Activity is given as a ratio of activity of GSNO-treated protein relative to activity of untreated protein. (B) β-amylase activity of untreated BAM3, BAM3 treated with 0.5 mM GSNO for 30 min, or BAM3 treated with GSNO


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Biochemistry

followed by 20 min treatment with either 5 mM DTT or 5 mM ascorbate prior to activity assay. (C) Western blot detection of protein glutathionylation. BAM3 (0.01 mg/mL) was incubated for 30 min either without (-) or with (+) 0.5 mM GSNO followed by denaturation and masking of unmodified cysteines with 40 mM iodoacetamide. Protein samples were concentrated and mixed with an equal volume of non-reducing Sample Loading Buffer. An equal amount of protein (0.5 ug) was run on a non-reducing SDS-PAGE gel and transferred to nitrocellulose for Western blotting with anti-GSH antibodies. Expected size for BAM3 is 59.9 kDa.

Identification of modified cysteines BAM3 has seven native cysteine residues, five of which are near the active site (Figure 3A). Three BAM3 cysteine residues, Cys169, Cys282 and Cys419, are conserved among all catalytic Arabidopsis BAMs whereas Cys177 and Cys213 are unique to BAM3 (Figure S2). BAM3 Cys257 and Cys433 are not widely conserved across Arabidopsis BAMs but homologous cysteines are present in few other Arabidopsis BAMs. We wanted to determine which cysteine(s) were being modified by GSNO to cause BAM3 inhibition so site-directed mutagenesis was used to generate mutants of BAM3 where each of these cysteines was replaced individually or in combinations. The cysteines were replaced either with a conservative serine (C169S, C213S, C282S, C419S, C433S) or with the residue in the homologous position in BAM1 (C177V and C257A). Recombinant mutant proteins were over-expressed and purified as described for wild-type BAMs. The β-amylase activity for each of the mutants was compared to wild type BAM3 to test whether the mutation itself impacted enzyme activity (Figure 3B). Replacement of the three conserved BAM3 cysteines, C169S, C282S, and C419S, resulted in almost complete loss of β-amylase


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activity, indicating that these cysteines are essential for β-amylase activity. BAM3 mutants lacking cysteines at the remaining four positions showed activity that was 50-80 % of wild-type BAM3.

Figure 3. Modeling and substitution of BAM3 native cysteines. (A) Homology model of BAM3 showing the location of all 7 native cysteine positions (space-filling) with an enlargement of the active site area. The mature BAM3 amino acid sequence (lacking the 85 amino acid predicted chloroplast transit peptide) was modeled onto soybean BAM5 crystal structures containing two maltose molecules (ball-and-stick) in the active site (PDB ID 1Q6C and 1WDR) using I-TASSER. (B) Specific activity of recombinant BAM3 with single cysteine-substitutions. The β-amylase


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Biochemistry

activity of Wild Type (WT) recombinant BAM3 was compared to BAM3 mutants containing single cysteine substitutions generated by site-directed mutagenesis. Each bar represents mean +/SD, n=3.

Cysteine-substitution mutants that showed greater than 50 % of wild-type BAM3 activity levels were then tested to determine the effect of pre-treatment with GSNO on β-amylase activity. If PTM of a given cysteine is responsible for the inhibition observed in BAM3 then the replacement of that cysteine would render the mutant less sensitive to GSNO treatment. As shown in Figure 4A, there was only one cysteine position, Cys433, where replacement generated an enzyme with significantly less GSNO sensitivity. When Cys433 was replaced with serine, the GSNO-treated enzyme had activity that was only slightly less than the untreated control. Western blots of GSNOtreated wild type and C433S BAM3 with anti-GSH antibodies showed that the glutathionylation detected in wild type BAM3 is absent in all BAM3 C433S mutants (Figure 4B).


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Figure 4. GSNO sensitivity and glutathionylation of BAM3 cysteine-substitution mutants. (A) The β-amylase activity of BAM3 cysteine-substitution mutants was measured following treatment with (black bars) or without (grey bars) 0.5 mM GSNO. Activity is given as a ratio of activity of GSNO-treated protein relative to activity of untreated protein. Each bar represents mean +/- SD, n=3. (B) Wild Type BAM3 and cysteine-substitution mutants treated with 0.5 mM GSNO as described in Figure 2C. Protein samples were run on a non-reducing SDS-PAGE gel and either stained with Coomassie Blue or blotted using anti-GSH antibodies as described in Experimental


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Procedures. Loading was normalized for equal protein concentrations. Separate boxes indicate separate gels and spaces indicate intervening lanes.

We sought to verify modification further by trypsin digesting GSNO-treated BAM3 and subjecting the peptide fragments to LC-ESI-TOF mass spectrometry (MS) to identify modified peptides. After GSNO treatment of recombinant BAM3, the protein was denatured and treated with the alkylating reagent iodoacetamide (IAM) to block any cysteines that were initially unmodified by GSNO. Analysis of MS data identified that the peptide containing Cys433 was present in both a glutathionylated (MW 2114.89, +305.06), and alkylated (MW 1866.82, +57) form (Figure 5A). This peptide (DGEQPEHANCSPEGLVK) contains a single cysteine residue, indicating that BAM3 is modified by glutathionylation of Cys433 in a portion of BAM3 proteins. Peptides containing cysteines 213, 257, 282 and 419 were also identified and did not show evidence of glutathionylation (Table S2). A cysteine-modified form of the peptide containing both cysteines 169 and 177 was not detected. According to the BAM3 homology structural model, Cys433 is located near the active site and would likely be solvent accessible for modification (Figure 3A) and this residue is highly conserved across BAM3 homologs (Figure S3). Modeling of a glutathione modification onto Cys433 did not significantly alter the BAM3 core backbone structure (Figure 5B), supporting the feasibility of this modification. Additionally, BAM3 Cys433 is predicted to be the most liable to glutathionylation within the BAM3 sequence by GSHsite, a web-based tool that predicts glutathionylation sites.52


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Figure 5. Glutathionylation of BAM3 Cys433. (A) Recombinant BAM3 was treated with 0.5 mM GSNO followed by 40 mM iodoacetamide (IAM) and then digested by trypsin as described in Experimental Procedures. Peptides were separated and analyzed by a HPLC-ESI-TOF mass spectrometer. Peptides covering ~80 % of total protein were identified. The mass spectra for peptide containing Cys433 (expected mass 1809.807 Da) is shown with arrows indicating peaks for IAM-alkylated (grey) and glutathionylated (GSH, black) forms of the peptide. m/z ratios and net peptide charges are listed above the peaks. Table S2 lists the expected and observed masses


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for all identified peptides containing cysteines. (B) Modeled structure showing glutathione (spacefilling) bonded to Cys433 of BAM3 (grey backbone) aligned with the unmodified BAM3 (blue backbone). (C) Modeled active site of BAM3 (grey surface view) containing substrate analog maltotetrose (yellow space-filling). (D) Modeled active site of BAM3 (grey surface view) containing glutathione (red space-filling) bonded to Cys433.

While testing the functional single cysteine substitution mutants of BAM3, we also tested double cysteine substitution mutants to determine if any of the double mutants could confer more complete insensitivity to GSNO than what was observed for the C433S mutant. None of the double mutants tested were more insensitive to GSNO than the C433S mutant (Figure 4A). However, we did observe that the C177V/C257A double mutant was less sensitive than either of the respective single mutants and appeared to diminish glutathionylation of BAM3 (Figure 4). These cysteines do not appear to be glutathionylated as the C433S substitution is sufficient to eliminate detection of glutathionylation and the triple cysteine mutant of BAM3, C177V/C257A/C433S, shows similar GSNO–sensitivity as the C433S mutant (Figure 4). Additionally, Cys257 glutathionylation was not detected by MS (Table S2). One possibility is that these cysteines may influence the ability of Cys433 to be modified.

BAM1 can be converted to GSNO-sensitive form As we were able to generate a largely GSNO-insensitive form of BAM3, we next sought to create a BAM1 that was sensitive to GSNO. BAM1 and BAM3 share about 50 percent sequence identity at the amino acid level. Indeed, BAM1 has a cysteine at position 454 that is homologous to the glutathionylated cysteine in BAM3 at 433. However, BAM1 activity was minimally affected


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by GSNO treatment (Figure 2A) even though anti-GSH blots indicate that the protein is being glutathionylated (Figure 6D). One way in which BAM1 and BAM3 differ is that BAM1 lacks cysteines in the positions homologous to BAM3’s Cys177 and Cys257. We saw that when these cysteines were removed from BAM3 the mutant protein showed less sensitivity to GSNO (Figure 4A) indicating that they may contribute to the GSNO-induced inhibition even though there is no evidence that these cysteines were glutathionylated in BAM3 (Figure 4B and Table S2). Given the apparent importance of these two cysteines, BAM1 single and double Cys-substitution mutants were generated by site-directed mutagenesis where cysteines were inserted to sites homologous to BAM3 Cys177 (BAM1 V197C) and Cys257 (BAM1 A277C). The BAM1 mutants retained ~50 % of the WT BAM1 activity (Figure 6A). Whereas the activity of the wild type BAM1 and the A277C mutant were minimally affected by GSNO treatment, we found that GSNO caused an inhibition of BAM1 V197C to about 40 % of control activity (Figure 6B), approaching the level of inhibition seen with GSNO-treated BAM3. The V197C/A277C double mutant was similarly inhibited in the presence of GSNO. This inhibition was reversed with DTT treatment but not ascorbate treatment suggesting inhibition was the result of glutathionylation (Figure 6C).50,51 So, the mutation of BAM1 V197C effectively switched the GSNO-responsiveness of BAM1 with that of BAM3.


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Figure 6. Specific activity and GSNO-sensitivity of BAM1 cysteine-substitution mutants. (A) The β-amylase activity of Wild Type (WT) recombinant BAM1 was compared to BAM1 containing single cysteine substitutions generated by site-directed mutagenesis. Each bar represents mean +/SD, n=3. (B) The β-amylase activity of BAM1 cysteine-substitution mutants was measured following treatment with (black bars) or without (grey bars) 0.5 mM GSNO. Activity is given as a ratio of activity of GSNO-treated protein relative to activity of untreated protein. Each bar represents mean +/- SD, n=3. (C) BAM1 V197C β-amylase activity was measured for untreated


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protein, protein treated with 0.5 mM GSNO for 30 min, or protein treated with GSNO followed by 20 min treatment with either 5 mM DTT or 5 mM ascorbate prior to activity assay as described in Figure 2B. (D) Wild type BAM1 was treated with (+) or without (-) 0.5 mM GSNO as described in Figure 2C. An equal amount of protein (0.5 ug) was run on a non-reducing SDS-PAGE gel and transferred to nitrocellulose for Western blotting with anti-GSH antibodies. Expected size for BAM1 is 64.6 kDa.

Discussion Our previous study revealed the intriguing disparity of a cold stress-induced increase in BAM3 transcript levels and decrease in BAM3 catalytic activity.13 In this study, we explored possible explanations for this observation. Western blots of leaf extracts showed nearly constant levels of BAM3 protein in cold stressed plants and in plants treated with SNP, indicating that the decrease in β-amylase activity is likely the result of enzyme inhibition (Figure 1B and D). As NO is induced by cold stress and released by SNP,17–19,53,54 the similar response of BAM3 in both cases suggests a possible role for NO in BAM3 inhibition. The fact that there was no large increase in BAM3 protein levels under cold stress, even though transcript levels increase, could mean that BAM3 transcripts are being stockpiled for increased translation during the recovery phase. Nakaminami et al. (2014) found that there are many genes upregulated during Arabidopsis cold stress that do not show a corresponding increase in protein levels. Instead these transcripts appear to be stored until temperatures return to normal and then are translated to aid in stress recovery.55 As the observed decrease in BAM3 activity appeared to be the result of enzyme regulation and NO-induced PTM is known to play an important role in cold stress signaling,56 we decided to explore the possibility that BAM3 was inhibited by nitrosylation or glutathionylation. Through in vitro assays with recombinant proteins, we showed that BAM3 is strongly and rapidly inhibited


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by GSNO treatment, the inhibition was reversible by reduction with DTT but not ascorbate, and inhibition coincided with the addition of at least one glutathionyl (GSH) group (Figure 2). Through site-directed mutagenesis and mass spectrometry, we determined that BAM3 Cys433 was being glutathionylated and that this PTM was responsible for inhibition (Figure 4 and 5A). Interestingly, Cys433 is highly conserved across BAM3 homologs in other eudicots (Figure S3) even though it is not essential for catalytic activity (Figure 3B). This cysteine is positioned near the active site and modeling indicates that a glutathionyl group at this position is structurally feasible and would block portions of the active site, thus interfering with starch binding (Figure 5B, C and D). Of additional interest is the potential involvement of Cys177 and Cys257 in modification of Cys433 and full inhibition of BAM3 by GSNO. Removal of these two cysteines by substitution resulted in a BAM3 mutant that was partially insensitive to GSNO with diminished BAM3 glutathionylation, although these cysteines do not appear to be glutathionylated themselves (Figure 4B, Table S2). One possibility is that these cysteines contribute to generating a favorable microenvironment for Cys433 glutathionylation, potentially by regulating access to Cys433 as both cysteines surround the active site (Figure 3A). The contribution of multiple cysteines towards inhibition was also supported by the creation of a GSNO-sensitive BAM1 mutant. Even though BAM1 has a cysteine homologous to BAM3 Cys433, it is not inhibited by GSNO in its wild type form. It was only when we generated a Cys-substitution mutant possessing a cysteine at the site homologous to BAM3 Cys177 (BAM1 V197C) that BAM1 become significantly inhibited by GSNO treatment, suggesting that either the cysteine at 197 was being modified or that it affected the sensitivity of Cys454 (Figure 6). Study of BAM1 and BAM3 has revealed multiple ways in which these similar proteins have differing enzymatic properties and regulation adapted to their unique roles. BAM3 is the principle


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β-amylase involved in leaf starch degradation at night and, although little is known about its regulation in vivo, recombinant protein showed feedback inhibition by maltotriose.10 BAM3 activity is optimal at lower temperatures and pH than BAM113 and is transcriptionally upregulated by cold.11 BAM1 has been shown to be transcriptionally upregulated by heat,11 catalytically activated in reducing environments,9 and be more stable at higher temperatures and pH,13 all of which are characteristics that are suited for BAM1’s primary function of breaking down starch in guard cells during the day. BAM1’s insensitivity to GSNO could be another important adaptation that allows this enzyme to function in guard cells during the day because NO is released regularly in guard cells where it plays a central role in stomatal closure.57,58 NO is established as an important factor in plant cold stress response, but less is known about the downstream targets of NO signaling in these conditions.17,19,59,60 High-throughput studies in plants have shown that NO-induction causes nitrosylation and glutathionylation of a number of chloroplast-localized proteins,17,29–32,61 although only a small number of these proteins have been studied in more detail to show whether glutathionylation alters function in vitro or in vivo. Alterations in starch metabolism, including accumulation of starch in leaves, are physiological responses to cold stress that could be targets for NO signaling.13,35–37 As BAM3 is the principle starch degradation enzyme in leaves, a possible model of regulation could be NO-mediated PTM of BAM3 under cold stress to limit starch breakdown. This is in agreement with our in vivo observations of decreased BAM3 activity under cold stress and NO release, and the in vitro GSNO inhibition of BAM3 by glutathionylation of a highly conserved cysteine near the active site. These findings indicate the need for further investigation into the involvement of BAM3 in cold stress response and how this protein fits into the larger mechanism of how plants acclimate and recover when exposed to changing temperature conditions.


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ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Supporting Information Document (PDF) containing the following: Table S1. Sequences of PCR primers used for QuikChange mutagenesis Table S2. HPLC-ESI-TOF-MS analysis of GSNO-treated and trypsin-digested BAM3 Figure S1. Purification of recombinant BAM1 and BAM3 protein Figure S2. Amino acid sequence alignment of the nine Arabidopsis thaliana β-amylase paralogs Figure S3. Alignment of partial BAM3 sequences showing Cys433 from ten eudicots

AUTHOR INFORMATION Corresponding Author * Department

of Biology, 951 Carrier Dr. MSC 7801, James Madison University, Harrisonburg,

VA 22807, phone 540-568-6649, email [email protected]. Present Addresses † Department of Biology, Western Carolina University, Cullowhee, NC 28723. Author Contributions


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ARS co-coordinated the study, designed, performed and analyzed the immunoblotting and MS experiments, supplied purified proteins, and wrote the paper. MRK performed and analyzed whole plant experiments. CEB provided technical assistance, performed protein modeling, and edited the manuscript. JDM conceived and co-coordinated the study, designed, performed and analyzed the enzyme assay experiments, and edited the paper. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by NSF RUI 1146776 and 1616467. ACKNOWLEDGMENT The authors would like to thank Dr. Samuel Zeeman and Dr. David Seung for generously supplying BAM3 antibodies and Dr. Heike Reinhold for BAM1 and BAM3 expression plasmids. Dr. Oleksandr Kokhan helped in advising and reviewing mass spectrometry data. Technical assistance was provided by Merwise Baray and Kelli Park.

ABBREVIATIONS The abbreviations used are: BAM, β-amylase; DTT, dithiothreitol; EB, extraction buffer; GSH, glutathione; GSNO, S-nitrosoglutathione; IAM, iodoacetamide; MS, mass spectrometry; NO, nitric oxide; PTM, post-translational modification; SNP, sodium nitroprusside; WT, wild type.

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For Table of Contents Use Only Glutathionylation inhibits catalytic activity of Arabidopsis β-amylase3 but not paralog β-amylase1 Amanda R. Storm, Matthew R. Kohler, Christopher E. Berndsen, Jonathan D. Monroe


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Glutathionylation inhibits catalytic activity of Arabidopsis Î²-amylase3

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