This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.
Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates. As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.
Glucan phosphatases are members of a functionally diverse subfamily of dual specificity phosphatases (DSPs) within the protein tyrosine phosphatase (PTP) superfamily1. They have been found in most life forms, including widely divergent photosynthetic organisms, humans, vertebrates, and some invertebrates and protists2,3,4. Plants contain three known glucan phosphatases: Starch Excess4 (SEX4), Like Sex Four1 (LSF1), and Like Sex Four2 (LSF2)5,6,7. Plants that lack glucan phosphatases display decreased rates of transitory starch degradation and accumulation of starch in the leaves8,9. Laforin is the founding member of the glucan phosphatase family that dephosphorylates glycogen in vertebrates and humans3,10. The mutations of laforin result in neurodegenerative Lafora disease, a fatal autosomal recessive form of epilepsy11. Glucan phosphatases are necessary for glycogen and starch metabolism and have emerged as important enzymes for modulating starch content in plants and treating neurodegenerative Lafora disease12,13. Recent X-ray crystallography studies on glucan phosphatases with model glucan substrates have shed light on substrate binding and the catalytic mechanism of glucan dephosphorylation14,15,16,17. However, the current understanding of how glucan phosphatases bind to their physiological substrates is incomplete.
Starch is an insoluble polymer of glucose made of 80%-90% amylopectin and 10%-20% amylose18. The substrates for plant glucan phosphatases are phosphorylated carbohydrate molecules, such as glycogen and starch granules. The phosphorylated glucosyl residues are present at a 1:600 phosphate:glucosyl residue ratio. Interestingly, the phosphates are present only on the amylopectin molecules19. The main plant glucan phosphatase SEX4 acts on the starch granule to dephosphorylate amylopectin molecules. The X-ray crystal structure of SEX4 combined with structure-guided mutagenesis studies has demonstrated the unique substrate specificities of SEX4 for different positions within a glucan structure15. We recently showed that the biologically relevant activity of SEX4 can only be observed when acting on its solubilized amylopectin substrates20. However, understanding glucan-SEX4 interactions has proven to be difficult due to the structural complexity of the substrate, broader binding specificities, and low binding affinities between the protein and its substrates. These issues have hindered the ability to utilize methods commonly used in protein-ligand interactions, such as isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy, and enzyme-linked immunosorbent assay (ELISA)-based assays.
Interestingly, much of our understanding of carbohydrate-protein interactions have come from studying lectins. Concanavalin A (ConA) is a legume lectin family of proteins originally extracted from the jack bean. ConA binds carbohydrates with high specificity, which is advantageous for its use in drug targeting and delivery applications. The binding of ConA to a variety of substrates containing nonreducing α-D-mannosyl and α-D-glucosyl has been extensively studied19,20. Commercially available ConA-bound Sepharose beads are commonly used to purify glycoproteins and glycolipids21. ConA binds to these glucans via C3, C4, and C6 hydroxyl groups of the glucose residues. ConA-Sepharose beads have also been successfully used to measure the binding of glycogen-protein and starch-protein interactions22,23. In this study, we used ConA-Sepharose beads to develop a binding assay to measure the binding specificities of glucan phosphatase-amylopectin interactions.
Previously, a ConA-based sedimentation assay was employed to assess glucan phosphatase substrate binding ability14,20,24. In this study, the same strategy was used to develop a novel method to determine the binding affinity of glucan-glucan phosphatase and carbohydrate interactions. This method also has an advantage for investigating various solubilized carbohydrate-protein interactions.
1. Preparation of ConA-Sepharose beads
2. Preparation of amylopectin solutions
3. Preparation of ConA-Sepharose: amylopectin beads
4. Incubating SEX4 with ConA-Sepharose:amylopectin beads
5. Running SDS-PAGE gels
6. Western blotting for chemiluminescence detection14,15
NOTE: This method can be easily modified/adapted depending on the western blotting equipment that users have in their labs.
7. Data analysis
Figure 1: Overview of the ConA-Sepharose sedimentation assay workflow. (A) Preparation of ConA-Sepharose beads. (B) Incubation with amylopectin substrate. (C) Incubation with SEX4 protein. (D) Separation of bound and unbound protein fractions through centrifugation. (E) Separation of protein through SDS-PAGE. (F) Western-blot analysis. (G) Chemiluminescence detection of His-tagged SEX4 protein. Please click here to view a larger version of this figure.
One of the key features of the glucan phosphatase family of proteins is their ability to bind to glucan substrates. First, the binding capacity of SEX4 to ConA-Sepharose:amylopectin beads was analyzed using SDS-PAGE (Figure 2A). Bovine serum albumin (BSA) served as a negative control to detect any nonspecific binding of proteins to the ConA-Sepharose:amylopectin beads. The SDS-PAGE analysis of proteins showed the presence of SEX4 protein in the pellet fraction and BSA in the supernatant fraction. W278A, a known SEX4 mutant with significantly reduced glucan binding ability, was also included in the assay. The W278A mutant appeared in the supernatant fraction, indicating the utility and specificity of this assay for detecting the glucan binding ability of SEX4 proteins. While SDS-PAGE visualized the SEX4 proteins, there was concern about the sensitivity and use of small glucan phosphatases; For instance, the ConA lectin protein could potentially interfere with detecting small proteins such as LSF2. To test whether this method could detect only the glucan phosphatases, a western blot was performed using an antibody specific to the N-terminal histidine tag. Indeed, there was a significant increase in the detection of SEX4, and the method is specific for detecting glucan phosphatases with an N-terminal histidine tag (Figure 2B). Quantitative measurement of ligand-protein interaction experiments requires performing essential controls for establishing the appropriate ligand and protein concentrations. Therefore, the co-sedimentation assay was tested next at three different SEX4 concentrations (Figure 2C–E). While 2 µg of SEX4 is sufficient to visualize via chemiluminescence detection, using 5 or 10 µg of SEX4 allows for a more accurate detection of the partial binding. The SEX4 binding against varying amylopectin concentrations (0.5 mg/mL, 1.0 mg/mL, and 5 mg/mL) was also tested. These results also indicate increased SEX4 binding with increased amylopectin concentration, with saturated binding at 5 mg/mL of amylopectin.
To determine the binding affinity of amylopectin and SEX4 wild-type protein, 10 µg of SEX4 was incubated with amylopectin (up to 5 mg/mL), and binding at each concentration was determined (Figure 3A). The fitting of percent protein-bound data to the specific, one-site binding model gave a KD of 1.03 ± 0.23 mg/mL (Figure 3B). There is no available crystal structure of SEX4 wild-type protein; instead, the crystal structure of catalytically inactive SEX4 C198S mutant reveals the binding interface of SEX4, made of DSP and carbohydrate-binding molecule (CBM) domains. The binding affinity of SEX4 C198S and amylopectin interaction was determined to have a KD of 0.11 ± 0.05 mg/mL for C198S mutant proteins; a ~10 times increased the binding affinity compared to SEX4 wild-type protein. While the exact mechanism of the increased binding of C198S is not understood, it is intriguing to see how a single-point mutation of SEX4 results in a significant increase in glucan binding ability.
Next, the applicability of this method to measure the binding of laforin, LSF2, corn (Zea mays) SEX4, and potato (Solanum tuberosum) SEX4 against potato amylopectin was assessed. Using the protocol presented here, the results demonstrated that laforin, LSF2, and SEX4 from agronomically important crops also differentially bind to amylopectin, suggesting different binding mechanisms for each protein (Figure 4A,B). Collectively, these findings revealed a successful method development for the determination of substrate binding of glucan phosphatase with amylopectin substrates.
Figure 2: Visualization of pellet (P) and supernatant (S) SEX4 fractions and optimization of the assay parameters. (A) Visualization of 5 µg of SEX4 protein (34 kDa) in the pellet and supernatant fractions using SDS-PAGE and Coomassie staining. The protein bands at 25 kDa and below are for the ConA protein attached to Sepharose beads. (B) Visualization of SEX4 proteins in the pellet and supernatant fractions via western analysis. 5 µg of SEX4 protein was detected via an antibody developed for the histidine tag. (C–E) The western analysis was done using varying amounts of SEX4 protein (2 µg, 5 µg, and 10 µg) and amylopectin concentrations (0.5 mg/mL, 1.0 mg/mL, and 5.0 mg/mL). A volume of 250 µL of beads and an incubation time of 30 min were used for all experiments to ensure equilibrium binding. Please click here to view a larger version of this figure.
Figure 3: Quantitative in vitro sedimentation assay. (A) Representative data of supernatant depletion and the gradual increase of pellet signal of SEX4 wild-type protein as the amylopectin concentration increased from 0 mg/mL to 5 mg/mL. (B) The percentage of SEX4 wild-type protein bound against varying amounts of amylopectin immobilized onto ConA-Sepharose beads. The beads were pelleted at 10,000 x g for 1 min, and both the supernatant and pellet proteins were separated by SDS-PAGE, detected by western analysis, and quantified to measure the percent bound. Data represent the average ± SD of three replicates. (C) Representative data of supernatant depletion and the gradual increase of pellet signal of SEX4 C198S protein as amylopectin concentration increased from 0 mg/mL to 5 mg/mL. (D) The percentage of SEX4 C198S protein bound against varying amounts of amylopectin immobilized onto ConA-Sepharose beads. The beads were pelleted at 10,000 x g for 1 min, and both the supernatant and pellet proteins were separated by SDS-PAGE, detected by western analysis, and quantified to measure the percent bound. Data represent the average ± SD of three replicates. Please click here to view a larger version of this figure.
Figure 4: Substrate binding of SEX4 orthologs, laforin, LSF2 against solubilized amylopectin. (A) Representative binding of each protein with 5 mg/mL of amylopectin immobilized in ConA-Sepharose beads. (B) The percentage of bound protein was calculated using the normalized chemiluminescence signals obtained for three independent experiments. Data represent the mean ± SD of three replicates. Please click here to view a larger version of this figure.
This study demonstrates the successful development of a novel in vitro sedimentation assay that allows determination of the binding affinity of glucan-glucan phosphatase interactions. The assay design takes advantage of the specific binding of lectin ConA to glucans via the hydroxyl residues of glucose to indirectly capture solubilized carbohydrate substrates onto Sepharose beads. This allows the separation of bound and unbound protein fractions via centrifugation and determination of the binding affinity of solubilized glucan substrates and glucan phosphatases. All of the glucan phosphatases tested were found to specifically bind to amylopectin, with no detectable binding to ConA-Sepharose beads in the absence of amylopectin.
The experiment was performed to measure the binding affinity with a small, fixed amount of SEX4 protein and a range of amylopectin concentrations attached to ConA-Sepharose beads. An excess of ConA-Sepharose beads was used to ensure the highest amylopectin concentration binding to beads. The complete binding of amylopectin was tested via a glucose assay. The protocol presented in the manuscript utilizes quantitative sedimentation to separate bound and unbound protein fractions, as well as gel electrophoresis and western analysis to measure the amount of SEX4 in the supernatant compared with the amount bound to the ConA-Sepharose:amylopectin beads. Quantifying the pellet fraction requires minimal washing to not disturb the binding equilibrium. To avoid any underestimation of pellet fraction, rather than examining the pellet, measuring the concentration of free SEX4 in the supernatant and calculating the difference of the bound SEX4:amylopectin complex is sufficient.
The in vitro sedimentation assay described here determined the differential binding affinity of SEX4 wild-type protein. Interestingly, the determined binding affinity of SEX4 wild-type protein using the developed in vitro sedimentation assay falls within the higher range of the reported value determined with a gel shift assay25. The binding assay should be sensitive to lower substrate concentrations and straightforward, whereas the gel shift assay requires making individual native gels in the presence of varying amounts of glucan substrates. This time-consuming method limits the number of concentrations that can be tested, and a limited substrate concentration range can be tested. Instead, sample preparation is fast and reliable with our in vitro sedimentation assay. Comparing SEX4 wild-type protein and SEX4 C198S mutant reveals a tenfold binding affinity difference. In this regard, this assay is sensitive to determine the binding of SEX4 mutant proteins quantitatively and qualitatively.
Protein-carbohydrate interactions are vital for many important biological processes, including catalysis, cell signaling, and recognition. They are also gaining increasing attention due to their important roles in the pharmaceutical and food industries. X-ray crystallography is widely used to obtain protein structures at the atomic level. However, the structural heterogeneity and flexibility of carbohydrate substrates make it harder to obtain carbohydrate-bound protein structures. Recently cryo-EM and nuclear magnetic resonance (NMR) spectroscopy have also been employed to determine the structures of carbohydrate-binding proteins26. Despite the many advancements in high-resolution structural techniques, it is challenging to obtain structures of carbohydrate-protein complexes. As a result, only a limited number of protein-carbohydrate complex structures have been solved experimentally. Furthermore, crystal structures only provide a snapshot of a protein at a certain conformation, and not the dynamic nature of substrate binding. Alternatively, several biophysical techniques, including isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR), are used to measure protein-carbohydrate interactions27. However, these methods are also not without limitations. One main challenge in determining protein-carbohydrate interactions is their relatively weak interactions compared to other protein-ligand interactions. The interactions within the range of high micromolar to low micromolar range significantly reduces the usability of ITC and SPR. Indirect methods, such as gel shift and pull-down assays, have gained significant attention for determining interactions that are difficult to assess by other methods25. Therefore, this binding assay is a widely applicable approach for sensitive measurements of protein and soluble carbohydrate interactions.
Glycogen and starch are major carbohydrate storage molecules made of branched polymers of glucose. The branching pattern, flexibility, and the presence of different microdomains within the starch and glycogen require interacting proteins to adopt unique substrate binding mechanisms. Much understanding of substrate recognition mechanisms of SEX4 has come from X-ray crystallographic studies coupled with structure-guided mutagenesis. To dephosphorylate starch, SEX4 must interact with significantly different microdomains of the starch granule to locate phosphates for position-specific dephosphorylation. Experimentally, it remains a challenge to determine the binding affinities of glucan phosphatases, given the weak interactions between the protein and the glucans and the structural flexibility and heterogeneity of carbohydrates. This manuscript presents a method to determine binding affinities of glucan phosphatase-carbohydrate interactions using a Concanavalin A (ConA)-based in vitro sedimentation assay. Glucan phosphatases employ distinct mechanisms to bind, locate, and dephosphorylate carbohydrates. Previously, ITC has been employed to measure the binding affinity of laforin and linear oligosaccharide chains. However, no study has revealed the binding affinities of glucan phosphatases with their physiological substrates via direct techniques such as ITC and SPR. To better understand the utility of this assay for determining substrate binding of glucan phosphatases, the binding of laforin, LSF2, and SEX4 against amylopectin was assessed. Future studies can be done to experimentally determine the binding affinity of all glucan phosphatases and their physiologically relevant glucan substrates. One of the critical steps of the method is to determine the optimum incubation times and substrate concentration range for the assay. Each glucan phosphatase binds differently with the substrate, and the method is optimized for SEX4. It is important to carry out initial optimization experiments to figure out these parameters. One other limitation is the use of histidine tagged protein for the assay. However, if antibodies are present for the study protein, the method can be easily optimized for any glucan-interacting protein.
The authors have nothing to disclose.
This study was supported by the National Science Foundation award MCB-2012074. The authors thank Dr. Craig W. Vander Kooi of the University of Florida Department of Biochemistry and Molecular Biology for valuable discussions and support. The authors also thank Dr. Matthew S. Gentry of the University of Florida Department of Biochemistry and Molecular Biology for his support. We would like to thank Dr. Sara Lagalwar, chair of the Skidmore College Neuroscience program, for allowing us to use the LICOR C-digit blot scanner for western blot imaging.
6x-His Tag monoclonal antibody (HIS.H8), HRP | Therm Fisher Scientific | MA1-21315-HRP | |
Biorad gel electrophoresis and Western blot kit | Biorad | 1703930 | |
Calcium chloride | Sigma-Aldrich | 208291 | |
C-Digit blot scanner | LICOR | 3600-00 | Blot scanner |
Complete protease inhibitor cocktail | Sigma-Aldrich | 11836170001 | |
Concanavalin A-sepharose beads | Sigma-Aldrich | C9017 | This product contains in 0.1 M acetate buffer, pH 6, containing 1 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2 in 20% ethanol |
Centrifuge | Eppendorf | 5425R | |
Glycine | Fisher Scientific | BP381-5 | |
GraphPad Prism 8.0 software | GraphPad | Version 8.0 | Data analysis software |
HEPES | Sigma-Aldrich | H8651 | |
Image Studio | LICOR | 3600-501 | Acquisition Software |
Magnesium chloride | Sigma-Aldrich | M2670 | |
Methanol | Fisher Scientific | A452SK-4 | |
Sodium dodecyl sulfate | Fisher Scientific | PI28312 | |
Potato amylopectin | Sigma-Aldrich | A8515 | |
Precast SDSPAGE Gels | Genscript | M00653S | |
Tris base | Fisher Scientific | BP154-1 | |
Tween 20 | Fisher Scientific | MP1TWEEN201 | |
Westernsure premium chemiluminescence substrate | LI-COR | 926-95000 |